Method for producing immunoglobulins containing protection proteins and their use

ABSTRACT

The immunoglobulins of the present invention are useful therapeutic immunoglobulins against mucosal pathogens such as  S. mutans . The immunoglobulins contain a protection protein that protects the immunoglobulins in the mucosal environment. 
     The invention also includes the greatly improved method of producing immunoglobulins in plants by producing the protection protein in the same cell as the other components of the immunoglobulins. The components of the immunoglobulin are assembled at a much improved efficiency. The method of the invention allows the assembly and high efficiency production of such complex molecules. 
     The invention also contemplates the production of immunoglobulins containing protection proteins in a variety of cells, including plant cells, that can be selected for useful additional properties. The use of immunoglobulins containing protection proteins as therapeutic antibodies against mucosal and other pathogens is also contemplated.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of co-pending application Ser. No. 08/434,000filed May 4, 1995, which is a continuation-in-part of co-pendingapplication Ser. No. 08/367,395 filed Dec. 30, 1994, each of which ishereby incorporated by reference in its entirety including drawings.

FIELD OF INVENTION

The present invention relates to expression of immunoglobulins in plantsthat contain a protection protein as well as to transgenic plants thatexpress such immunoglobulins. The therapeutic use of theseimmunoglobulins is also contemplated.

BACKGROUND TO THE INVENTION

Monoclonal antibodies have great potential for numerous therapeuticpurposes. The advantages of monoclonal antibody therapeutics overconventional pharmaceuticals include their exquisite selectivity,multiple effector functions, and ease of molecular manipulation such asradioisotope labelling and other types of conjugation. A wide variety oftarget antigens have been used to generate specific monoclonalantibodies. See for example Therapeutic Monoclonal Antibodies, C. A. K.Borrebaeck and J. W. Larrick eds., Stockton Press, New York, 1990, andThe Pharmacology of Monoclonal Antibodies, M. Rosenberg and G. P. Mooreeds., Springer-Verlag, Berlin, 1994.

One therapeutic application of monoclonal antibodies is passiveimmunotherapy in which the exogenously produced immunoglobulins areadministered directly to the animal being treated by injection or byingestion. To be successful, passive immunotherapy must deliver anappropriate amount of an immunoglobulin to the animal, because passiveimmunotherapy does not rely on an immune response in the animal beingtreated. The immunoglobulins administered must be specific for thepathogen or molecule desired to effect treatment. One advantage ofpassive immunotherapy is the speed at which the antibody can becontacted with the target compared to a normal immune response. Passiveimmunotherapy can also be used as a prophylaxis to prevent the onset ofdiseases or infections.

A major potential use of passive immunotherapy is in combating bacterialinfections. Recent emergence of anti-biotic resistant bacteria maketreatment of bacterial infections with passive immunotherapy desirable.Antibiotic treatment targeted to a single pathogen often involveseradication of a large population of normal microbes, and this can haveundesired side effects. An alternative approach has been to utilize theinherent specificity of immunoglobulins to inhibit a specific pathogenicfunction in very specific microbial populations. In this strategy,purified immunoglobulins of the appropriate specificity would beadministered in order to provide a passive barrier to pathogen invasion.

In addition, the immunoglobulins used for passive immunotherapies forexample, for oral administration of immunoglobulins must meet certainrequirements. First, the immunoglobulin must be functional in very harshenvironments, such as the gastrointestinal tract. Second, theimmunoglobulin must be resistant to the actions of proteases so that itwill not be degraded prior to inactivating the target.

Certain types of cells, including epithelial cells and hepatocytes, arecapable of assembling immunoglobulin molecules which have beenspecifically adapted to function in harsh environments. Theseimmunoglobulins are referred to as secretory immunoglobulins (SIg) andinclude both secretory IgA (SIgA) and secretory IgM (SIgM). Theprotection provided by endogenous secretory immunoglobulins have beendemonstrated. Several mechanisms for protection from bacterial infectionby secretory immunoglobulins have been proposed, including, but notlimited to, direct killing, agglutination, inhibition of epithelialattachment and invasion, inactivation of enzymes and toxins,opsonization and complement activation. In an animal, endogenouslyproduced SIgA are exposed to very harsh environments where numerousproteases, such as intestinal and bacterial enzymes are extremely activeand denaturants, such as stomach acid, are also present.

One component of secretory immunoglobulins, the secretory component,helps to protect the immunoglobulin against these inactivating agentsthereby increasing the biological effectiveness of secretoryimmunoglobulin.

The mechanism of synthesis and assembly of these secretoryimmunoglobulins, such as SIgA or SIgM is extremely complex. In animalcells, secretory immunoglobulins are assembled in a process involvingdifferent cell types. Each secretory immunoglobulin is made up ofimmunoglobulin heavy and light chains, joining chain (J chain) and asecretory component. The immunoglobulin producing B cells make andassemble the immunoglobulin heavy and light chain together with J chainto produce dimeric or polymeric IgM or IgA. The secretory component isproduced by a second type of cell, either epithelial cells orhepatocytes, and secretory immunoglobulin is assembled in and secretedfrom these cells. The mechanism by which these cells assemble andsecrete the secretory immunoglobulin is extremely complex and requires aunique microenvironment provided, for example, by mucosal tissues. Themicroenvironment places the B cells that produce the polymericimmunoglobulin near the cells that assemble and secrete secretoryimmunoglobulin onto the mucosal surface of an animal.

The epithelial cells have a receptor, the polyimmunoglobulin receptor(pIgR), that specifically recognizes and binds polymericimmunoglobulin/containing J chain, internalizing it and transporting itthrough the epithelial cell. Expressed on the basolateral cell surface,the pIgR has an N-terminal signal peptide of 18 amino acids, anextracellular polyimmunoglobulin binding portion of 629 amino acids, amembrane spanning segment of 23 hydrophobic residues, and a cytoplasmictail of 103 amino acids. The extra-cellular portion contains fiveimmunoglobulin-like domains of 100-111 amino acids each and constitutesthe secreted form of the molecule. See for example, Mostov, Ann. Rev.Immol., 12:63-84 (1994) The site at which the polyimmunoglobulinreceptor is cleaved to generate mature secretory component has not beenaccurately determined.

The polyimmunoglobulin receptor is located on the basolateral surface ofepithelial cells in animals. Polymeric J chain-containingimmunoglobulins produced in B cells interact with and are bound by thereceptor resulting in vesicularization, transport across the epithelialcell, and ultimate secretion to the mucosal surface. Transepithelialtransport also involves proteolysis and phosphorylation to produce themature SIg containing the secretory component. The close association ofthe required cells found in the mucosal microenvironment, specificallythe B lymphocytes and epithelial cells, is required for secretoryimmunoglobulin assembly.

The targeting of the production of immunoglobulins in transgenicorganisms, such as mice, is extremely difficult and transgenic organismsmade from fungus or plants do not contain the proper cell types andmucosal microenvironment to produce secretory immunoglobulins. Theproduction of large amounts of secretory immunoglobulins in transgenicorganisms and cell culture has, before this invention, been impossible.One desiring to produce a secretory immunoglobulin in cell culture or atransgenic organism must express the immunoglobulin heavy chain, theimmunoglobulin light chain, and J chain in a B lymphocyte. To mimic theproper mucosal microenvironment a cell having the pIgR receptor on itssurface would also have to be present and be in close association withthat B lymphocyte to even attempt to assemble a functional secretoryimmunoglobulin.

This elaborate process required for natural secretory immunoglobulinassembly is extremely difficult to duplicate in cell culture ortransgenic organisms. Production of SIg in cell culture or transgenicorganisms would require coupling the functions of cells producingimmunoglobulin with the functions of epithelial cells in artificial (invitro) systems. Moreover, if the desired transgenic organism is afungus, a bacterium, or a plant, the cell types and pathways ofreceptor-mediated cellular internalization, transcytosis, and secretionsimply are not present. Those organisms lack epithelial cells and therequired mucosal microenvironment.

To date only the assembly of immunoglobulins having light, heavy and Jchain within the same cell has been reported. See Carayannopoulos et al.Proc. Nat. Acad. Sci. U.S.A., 91:8348-8352 (1994). However, the assemblyof an immunoglobulin having the additional protein component, secretorycomponent, within a single cell has not been described.

The present invention discloses a novel method for the assembly of thesecomplex molecules. Rather than assemble the tetrameric complex at theepithelial cell surface by the interaction of a membrane boundpolyimmunoglobulin receptor with immunoglobulin, we have assembledsecretory immunoglobulin composed of alpha, J, and kappa immunoglobulinchains associated with a protection protein derived from pIgR. Thisinvention produces transgenic plants that assemble secretoryimmunoglobulins with great efficiency. The present invention makespassive immunotherapy economically feasible.

SUMMARY OF THE INVENTION

The present invention contemplates a new type of immunoglobulinmolecule. Immunoglobulins of the present invention contain a protectionprotein in association with an immunoglobulin derived heavy chain havingat least a portion of an antigen binding domain. In other embodiments,the immunoglobulin of the present invention further comprise animmunoglobulin derived light chain having at least a portion of anantigen binding domain associated with the immunoglobulin derived heavychain.

The protection proteins of the present invention give theimmunoglobulins containing these protein useful properties includingresistance to chemical and enzymatic degradation and resistance todenaturation. These protection proteins enhanced the resistance of theimmunoglobulins to environmental conditions.

The protection proteins of the proteins of the present inventioncomprise at least a segment of amino acid residues 1 to 606 of nativepolyimmunoglobulin receptor (pIgR) of any species. Other usefulprotection proteins include protection proteins that contain portions ofthe pIgR molecule. For example, the protection protein may comprise allor part of: amino acids 1-118 (domain I of rabbit pIgR), amino acids 1to 223 (domains I and II of rabbit pIgR); amino acids 1 to 332 (domainsI, II, III of rabbit pIgR); amino acids 1 to 441 (domains I, II, III,and IV rabbit of pIgR); amino acids 1 to 552 (domains I, II, III, IV andV of rabbit pIgR); and amino acids 1 to 606 or 1 to 627 of pIgR.Additional amino acids, derived either from the pIgR sequence 653-755,or from other sources, may be included so long as they do not constitutea functional transmembrane spanning segment.

In other preferred embodiments, the immunoglobulins of the presentinvention have a protection protein which has a first amino acidsequence which substantially corresponds to at least a portion of theamino acid residues 1 to 606 or 1 to 627 of the rabbitpolyimmunoglobulin receptor and has a second amino acid residue sequencecontiguous with said first amino acid sequence, wherein said secondamino acid residue sequence does not have an amino acid residue sequencecorresponding to the transmembrane segment of the rabbitpolyimmunoglobulin receptor.

In more preferred embodiments, the second amino acid residue sequencehas at least a portion of an amino acid sequence which corresponds toamino acid residues 655 to 755 of a polyimmunoglobulin receptor. Inother preferred embodiments, the second amino acid residue is at least aportion of one or more of the following: an intracellular domain of apolyimmunoglobulin molecule, a domain of a member of the immunoglobulingene superfamily, an enzyme, a toxin, or a linker.

The present invention contemplates protection proteins which do not havean amino acid residue corresponding to the transmembrane segment ofrabbit polyimmunoglobulin receptor but may have amino acid residuescorresponding to the intracellular domain of the rabbitpolyimmunoglobulin receptor and this are deletion mutants of thereceptor.

The present invention also contemplates immunoglobulins containingprotection proteins which have an amino acid sequence which does notcontain amino acid residues of a polyimmunoglobulin receptor from aspecies which are analogous to amino acid residues 288 to 755 of therabbit immunoglobulin receptor, but does contain at least a portion ofthe amino acid residues or the domains from a polyimmunoglobulinreceptor of a species which are analogous to one or more of these aminoacid segments: Amino acids corresponding to amino acid residues 20-45 ofthe rabbit polyimmunoglobulin receptor; amino acids corresponding to oranalogous to amino acid residues 1 to 120 of the rabbitpolyimmunoglobulin receptor: amino acids corresponding to or analogousto amino acid residues numbers 120-230 of the rabbit immunoglobulinreceptor; amino acids corresponding to or analogous to amino acidresidues numbers 230-340 of the rabbit polyimmunoglobulin receptor;amino acids corresponding to or analogous to amino acid residues 340-456of the rabbit polyimmunoglobulin receptor; amino acids corresponding toor analogous to amino acid residues numbers 450-550 to 570 of the rabbitpolyimmunoglobulin receptors; amino acids corresponding to or analogousto amino acid residues 550 to 570-606 to 627 of the rabbitpolyimmunoglobulin receptor.

The protection proteins of the present invention may be derived frommany species and include protection proteins derived from mammals,rodents, humans, bovine, porcine, ovine, fowl, caprine, mouse, rat,guinea pig, chicken or other bird and rabbit.

In preferred embodiments, the immunoglobulins of the present inventioncontain two or four immunoglobulin derived heavy chains having at leasta portion of an antigen binding domain associated with the protectionprotein and two or four immunoglobulin derived light chains having atleast a portion of an antigen binding domain bound to the each of theimmunoglobulin derived heavy chains.

In other preferred embodiments, the immunoglobulins of the presentinvention further comprise immunoglobulin J chain bound to at least oneof the immunoglobulin derived heavy chains. In preferred embodiments,the component parts of the immunoglobulins of the present invention arebound together by hydrogen bonds, disulfide bonds, covalent bonds, ionicinteractions or combinations of said bonds. In other preferredembodiments, the immunoglobulin of the present invention containprotection proteins and/or immunoglobulin derived heavy, light or Jchains that are free from N-linked and/or O-linked oligosaccharides.

The immunoglobulins of the present invention may be used as therapeuticimmunoglobulins against, for example, mucosal pathogen antigens. Inpreferred embodiments, the immunoglobulins of the present invention arecapable of preventing dental caries by binding to an antigen from S.mutans serotypes c, e and f; and S. sobrinus stereotype d and g, usingolder nomenclature S. mutans a, c, d, e, f, g and h.

The present invention also contemplates a eukaryotic cell, including aplant cell, containing an immunoglobulin of the present invention.Eukaryotic cells, including plant cells, containing a nucleotidesequence encoding a protection protein and a nucleotide sequenceencoding an immunoglobulin derived heavy chain having at least a portionof an antigen binding domain is also contemplated. Eukaryotic cells,including plant cells, that additionally contain a nucleotide sequenceencoding an immunoglobulin derived light chain having at least a portionof an antigen binding domain is also contemplated. In preferredembodiments, the eukaryotic cells, including plant cells, of the presentinvention contain nucleotide sequences that encode immunoglobulins thathave an antigen binding domain is capable of binding an antigen from S.mutans serotypes a, c, d, e, f, and g, h (S. mutans serotypes c, e and fand S. sobrinus serotypes d and g under new nomenclature. The nucleotidesequences include RNA and appropriate DNA molecules arranged forexpression.

In preferred embodiments, the plant cells of the present invention arepart of a plant such as a whole plant. The present inventioncontemplates the use of all types of plants, both dicotyledonous andmonocotyledonous including alfalfa, and tobacco.

The present invention also contemplates compositions comprising animmunoglobulin of the present invention and plant macromolecules derivedfrom one of the plants useful in practicing the present invention.Particularly contemplated are compositions containing ribulosebisphosphate carboxylase, light harvesting complex, pigments, secondarymetabolites or chlorophyll and an immunoglobulin of the presentinvention. Preferred compositions have an immunoglobulin concentrationof between 0.001% and 99.9% mass excluding water. In more preferredembodiments, the immunoglobulin concentrations present in thecomposition is between 0.1% and 99%. Other preferred compositions haveplant macromolecules present in a concentration of between 1% and 99%mass excluding water.

The present invention also contemplates methods for making animmunoglobulin of the present invention comprising introducing into aplant cell an expression vector having a nucleotide sequence encoding aprotection protein operably linked to a transcriptional promoter; andintroducing into the same plant cell an expression vector containing anucleotide sequence encoding an immunoglobulin derived heavy chainhaving at least a portion of an antigen binding domain, operably linkedto a transcriptional promoter. Other methods that further include thestep of introducing into the same plant cell an expression vectorcontaining a nucleotide sequence encoding an immunoglobulin derivedlight chain having at least a portion of an antigen binding domain,operably linked to a transcriptional promoter. Other preferred methodsinclude also introducing into a plant cell an expression vectorcontaining a nucleotide sequence encoding an immunoglobulin J chainoperably linked to a transcriptional promoter.

The present invention also contemplates methods for producing assembledimmunoglobulins having heavy, light and J chains and a protectionprotein by introducing into a eukaryotic cell nucleotide sequencesoperatively linked for expression to encode an immunoglobulin derivedheavy chain having at least a portion of an antigen binding domain, animmunoglobulin light chain having at least a portion of an antigenbinding domain, and immunoglobulin J chain, and a protection protein.The method further comprises maintaining the eukaryotic cell underconditions allowing the production and assembly of the immunoglobulinderived heavy and light chains together with the immunoglobulin J chainand the protection protein to form an immunoglobulin containing aprotection protein.

The present invention also contemplates methods of making animmunoglobulin resistant to various environmental conditions (morestable) and harsh conditions by operatively linking a nucleotidesequence encoding at least a portion of a desirable antigen bindingdomain derived from an immunoglobulin heavy chain to a nucleotidesequence encoding at least one domain derived from an immunoglobulin μor α (IgM or IgA) heavy chain (or other immunoglobulin having increasedstability in the environment) to form a nucleotide sequence encoding achimeric immunoglobulin heavy chain and expressing that nucleotidesequence in a eukaryotic which also contains at least one molecule fromthe following list: a protection protein, an immunoglobulin derivedlight chain having at least a portion of an antigen binding domain andan immunoglobulin J chain. The method further comprises allowing thechimeric immunoglobulin heavy chain to assemble with the other moleculepresent in the same cell to form an immunoglobulin which is resistant toenvironmental conditions and more stable.

The large scale production of immunoglobulins of the present inventionis contemplated by growing the plants of the present invention andextracting the immunoglobulins from those plants. In preferredembodiments, the method of producing therapeutic immunoglobulincompositions containing plant macromolecules includes the step ofshearing under pressure a portion of a plant of the present invention toproduce a pulp containing a therapeutic immunoglobulin and plantmacromolecules in an liquid derived from the apoplast or symplast of theplant and solid plant derived material. Further processing steps arecontemplated which include separating the solid plant derived materialfrom the liquid and using a portion of the plant including a leaf, stem,root, tuber, flower, fruit, seed or entire plant. The present inventioncontemplates the use of a mechanical device or enzymatic method whichreleases liquid from the apoplast or symplast of said plant followedoptionally by separating using centrifugation, settling, flocculation orfiltration.

The present invention contemplates immunoglobulins that are chimeric andthus they contain immunoglobulin domains derived from differentimmunoglobulin molecules. Particularly preferred are immunoglobulinscontaining domains from IgG, IgM and IgA.

The present invention contemplates immunoglobulins where theimmunoglobulin derived heavy chain is comprised of immunoglobulindomains from two different isotopes of immunoglobulin. In preferredembodiments, the immunoglobulin domains used include at least theC_(H)1, C_(H)2, or C_(H)3 domain of mouse IgG, IgG1, IgG2a, IgG2b, IgG3,IgA, IgE, or IgD or the Cvar domain. In other preferred embodiments, theimmunoglobulin heavy chain is comprised of at least the Cμ1, Cμ2, Cμ3 orCμ4 domain of mouse IgM.

The present invention also contemplates immunoglobulin derived heavychains made up of immunoglobulin domains include at least the C_(H)1,C_(H)2, or C_(H)3 domain of a human IgG, IgG1, IgG2, IgG3, IgG4, IgA1,IgA2, or IgD; or least the Cμ1, Cμ2, Cμ3 or Cμ4 domain of human IgM; orthe Cvar domain. The use of immunoglobulin domains derived from mammals,animals or rodents including any IgG isotype, any IgA isotype, IgE, IgMor IgD is contemplated.

The present invention also contemplates tetratransgenic organisms whichare comprised of cells containing four different transgenes eachencoding a different polypeptide of a multipeptide molecule wherein atleast one of those peptides is associated together to form amultipeptide molecule. The transgenic organisms contemplated by thepresent invention include transgenic organisms which contain as one ofthe four transgenes present a transgene encoding a protection protein.The protection protein present in the transgenic organism's cells isable to assemble together with immunoglobulin heavy chains when presentto form immunoglobulins which contain the protection protein.

In preferred transgenic organisms, the cells of the organism expressfour transgenes which encode an immunoglobulin derived heavy chainhaving at least a portion of an antigen binding domain, animmunoglobulin derived light chain having at least a portion of anantigen binding domain, an immunoglobulin J chain, and a protectionprotein. In other preferred transgenic organisms, the cells contain atransgene which encodes a chimeric immunoglobulin heavy chain, animmunoglobulin heavy chain derived form an IgA heavy chain, animmunoglobulin derived from an IgM heavy chain or an immunoglobulinderived from some other isotype of heavy chain.

In the most preferred embodiment, the transgenic organisms of thepresent invention are a plant. Various types and species of plants arecontemplated by the present invention. In addition, the presentinvention also contemplates mammals which are transgenic organismscontaining the various molecules of the present invention. Mammaliantransgenic organisms are contemplated by the present invention andinclude mammalian transgenic organisms which contain four transgenesencoding different polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings will first briefly be described.

FIG. 1 illustrates synthetic oligonucleotides J1-J5 (restriction enzymesites are underlined) that were used to amplify DNA fragments for Guy's13 and alpha chain domains in the construction of hybrid IgG/A heavychains. The relative positions of the areas encoded by eacholigonucleotide are shown diagrammatically The resulting recombinantheavy chains produced by combining various DNA fragments expressed inplants are also shown.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Dicotyledon (dicot): A flowering plant whose embryos have two seedhalves or cotyledons. Examples of dicots are: tobacco; tomato; thelegumes including alfalfa; oaks; maples; roses; mints; squashes;daisies; walnuts; cacti; violets; and buttercups.

Monocotyledon (monocot): A flowering plant whose embryos have onecotyledon or seed leaf. Examples of monocots are: lilies; grasses; corn;grains, including oats, wheat and barley; orchids; irises; onions andpalms.

Lower plant: Any non-flowering plant including ferns, gymnosperms,conifers, horsetails, club mosses, liver warts, hornworts, mosses, redalgaes, brown algaes, gametophytes, sporophytes of pteridophytes, andgreen algaes.

Eukaryotic hybrid vector: A DNA by means of which a DNA coding for apolypeptide (insert) can be introduced into a eukaryotic cell.

Extrachromosomal ribosomal DNA (rDNA): A DNA found in unicellulareukaryotes outside the chromosomes, carrying one or more genes codingfor ribosomal RNA and replicating autonomously (independent of thereplication of the chromosomes).

Palindromic DNA: A DNA sequence with one or more centers of symmetry.

DNA: Deoxyribonucleic acid.

T-DNA: A segment of transferred DNA.

rDNA: Ribosomal DNA.

RNA: Ribonucleic acid.

rRNA: Ribosomal RNA.

Ti-plasmid: Tumor-inducing plasmid.

Ti-DNA: A segment of DNA from Ti-plasmid.

Insert: A DNA sequence foreign to the rDNA, consisting of a structuralgene and optionally additional DNA sequences.

Structural gene: A gene coding for a polypeptide and being equipped witha suitable promoter, termination sequence and optionally otherregulatory DNA sequences, and having a correct reading frame.

Signal Sequence: A DNA sequence coding for an amino acid sequenceattached to the polypeptide which binds the polypeptide to theendoplasmic reticulum and is essential for protein secretion.

(Selective) Genetic marker: A DNA sequence coding for a phenotypicaltrait by means of which transformed cells can be selected fromuntransformed cells.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a gene and to which RNApolymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Inducible promoter: A promoter where the rate of RNA polymerase bindingand initiation is modulated by external stimuli. Such stimuli includelight, heat, anaerobic stress, alteration in nutrient conditions,presence or absence of a metabolite, presence of a ligand, microbialattack, wounding and the like.

Viral promoter: A promoter with a DNA sequence substantially similar tothe promoter found at the 5′ end of a viral gene. A typical viralpromoter is found at the 5′ end of the gene coding for the p21 proteinof MMTV described by Huang et al., Cell, 27:245 (1981). Other examplesinclude the promoters found in the 35S transcript of the cauliflowermosaic virus as described by Benfey et al., Science, 250:959 (1990).

Synthetic promoter: A promoter that was chemically synthesized ratherthan biologically derived. Usually synthetic promoters incorporatesequence changes that optimize the efficiency of RNA polymeraseinitiation.

Constitutive promoter: A promoter where the rate of RNA polymerasebinding and initiation is approximately constant and relativelyindependent of external stimuli. Examples of constitutive promotersinclude the cauliflower mosaic virus 35S and 19S promoters described byPoszkowski et al., EMBO J., 3:2719 (1989) and Odell et al., Nature,313:810 (1985).

Regulated promoter: A promoter where the rate of RNA polymerase bindingand initiation is modulated at a specific time during development, or ina specific structure of an organism or both of these types ofmodulation. Examples of regulated promoters are given in Chua et al.,Science, 244:174-181 (1989).

Single-chain antigen-binding protein: A polypeptide composed of animmunoglobulin light-chain variable region amino acid sequence (V_(L))tethered to an immunoglobulin heavy-chain variable region amino acidsequence (V_(H)) by a peptide that links the carboxyl terminus of theV_(L) sequence to the amino terminus of the V_(H) sequence. Generallyany combination of the heavy chain and light chain antigen bindingdomains into the same polypeptide using a linker polypeptide to allowthe binding domains to assume a useful conformation. Such combinationsinclude V_(H)-Linker-V_(L), V_(H)-Linear-Light chain, orV_(L)-Linear-Fd.

Single-chain antigen-binding protein-coding gene: A recombinant genecoding for a single-chain antigen-binding protein.

Polypeptide and peptide: A linear series of amino acid residuesconnected one to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues.

Protein: A linear series of greater than about 50 amino acid residuesconnected one to the other as in a polypeptide.

Immunoglobulin product: A polypeptide, protein or protein containing atleast the immunologically active portion of an immunoglobulin heavychain and is thus capable of specifically combining with an antigen.Exemplary immunoglobulin products are an immunoglobulin heavy chain,immunoglobulin molecules, substantially intact immunoglobulin molecules,any portion of an immunoglobulin that contains the paratope, includingthose portions known in the art as Fab fragments, Fab′ fragment, F(ab′)₂fragment and Fv fragment.

Immunoglobulin molecule: A protein containing the immunologically activeportions of an immunoglobulin heavy chain and immunoglobulin light chaincovalently coupled together and capable of specifically combining withantigen.

Immunoglobulin derived heavy chain: A polypeptide that contains at leasta portion of the antigen binding domain of an immunoglobulin and atleast a portion of a variable region of an immunoglobulin heavy chain orat least a portion of a constant region of an immunoglobulin heavychain. Thus, the immunoglobulin derived heavy chain has significantregions of amino acid sequence homology with a member of theimmunoglobulin gene superfamily. For example, the heavy chain in an Fabfragment is an immunoglobulin derived heavy chain.

Immunoglobulin derived light chain: A polypeptide that contains at leasta portion of the antigen binding domain of an immunoglobulin and atleast a portion of the variable region or at least a portion of aconstant region of an immunoglobulin light chain. Thus, theimmunoglobulin derived light chain has significant regions of amino acidhomology with a member of the immunoglobulin gene superfamily.

Antigen binding domain: The portion of an immunoglobulin polypeptidethat specifically binds to the antigen. This antigen is typically boundby antigen binding domains of the immunoglobulin heavy and light chain.However, antigen binding domains may be present on a single polypeptide.

J chain: Is a polypeptide that is involved in the polymerization ofimmunoglobulins and transport of polymerized immunoglobulins throughepithelial cells. See, The Immunoglobulin Helper: The J Chain inImmunoglobulin Genes, at pg. 345, Academic Press (1989). J chain isfound in petameric IgM and dimeric IgA and typically attached viadisulphide bonds. J chain has been studied in both mouse and human.

Fab fragment: A protein consisting of the portion of an immunoglobulinmolecule containing the immunologically active portions of animmunoglobulin heavy chain and an immunoglobulin light chain covalentlycoupled together and capable of specifically combining with antigen. Fabfragments are typically prepared by proteolytic digestion ofsubstantially intact immunoglobulin molecules with papain using methodsthat are well known in the art. However an Fab fragment may also beprepared by expressing in a suitable host cell the desired portions ofimmunoglobulin heavy chain and immunoglobulin light chain using methodswell known in the art.

F_(v) fragment: A protein consisting of the immunologically activeportions of an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region covalently coupled togetherand capable of specifically combining with antigen. F_(v) fragments aretypically prepared by expressing in suitable host cell the desiredportions of immunoglobulin heavy chain variable region andimmunoglobulin light chain variable region using methods well known inthe art.

Asexual propagation: Producing progeny by regenerating an entire plantfrom leaf cuttings, stem cuttings, root cuttings, single plant cells(protoplasts) or callus.

Self-pollination: The transfer of pollen from male flower parts tofemale flower parts on the same plant. This process typically producesseed.

Cross-pollination: The transfer of pollen from the male flower parts ofone plant to the female flower parts of another plant. This processtypically produces seed from which viable progeny can be grown.

Epitope: A portion of a molecule that is specifically recognized by animmunoglobulin product. It is also referred to as the determinant orantigenic determinant.

Chimeric immunoglobulin heavy chain: An immunoglobulin derived heavychain having at least a portion of its amino acid sequence derived froman immunoglobulin heavy chain of a different isotype or subtype or someother peptide, polypeptide or protein. Typically, a chimericimmunoglobulin heavy chain has its amino acid residue sequence derivedfrom at least two different isotypes or subtypes of immunoglobulin heavychain.

Transgene: A gene that has been introduced into the germ line of ananimal. The gene may be introduced into the animal at an earlydevelopmental stage. However, the gene could be introduced into thecells of an animal at a later stage by, for example, a retroviralvector.

Multiple molecule: A molecule comprised of more than one peptide orpolypeptide associated together by any means including chemical bonds.

B. Immunoglobulins Containing Protection Proteins

The present invention provides novel methods for producingimmunoglobulin molecules containing protection proteins. Theimmunoglobulins contain a protection protein in association with animmunoglobulin derived heavy chain that has at least a portion of anantigen binding domain.

The protection proteins of the present invention have an amino acidsequence substantially corresponding to or analogous to at least aportion of residues 1 to 627 of the amino acid residue sequence of therabbit polyimmunoglobulin receptor and is derived from a precursorprotein that does not contain the amino acid residue sequence greaterthan amino acid residue 627 or analogous to amino acid residue 627 ofthe rabbit polyimmunoglobulin receptor. The nucleotide sequence and theamino acid sequence of the rabbit polyimmunoglobulin receptor are nowand have been described by the Mostov et al., Nature, 308:37 (1984) andEMBL/Gene Bank K01291. The nucleotide sequence of the polyimmunoglobulinreceptor is SEQ ID NO. 1 and the corresponding amino acid residuesequence is SEQ ID NO. 2.

The polyimmunoglobulin receptors from any species may be used as aprotection protein and these protection proteins do not contain and arederived from a precursor protein that does not contain amino acidshaving numbers greater than the amino acid number analogous to aminoacids 1-627 of the rabbit immunoglobulin sequence. In preferredembodiments, the protection protein is derived from any species andprecursor protein that contains amino acids analogous to at least aportion of amino acids 1-606 of the rabbit polyimmunoglobulin receptorand does not contain amino acid residues analogous to residues 607-755of the rabbit polyimmunoglobulin receptor.

The human polyimmunoglobulin receptor sequence has been determined andreported by Krajci et al., Eur. J. Immunol., 22:2309-2315 (1992) andKrajci et al., Biochem. Biophys. Res. Comm., 158:783-789 (1989) andEMBL/Gene Bank Accession No. X73079. The nucleotide sequence of thehuman polyimmunoglobulin receptor is SEQ ID NO. 3 and the correspondingamino acid residue sequence is. SEQ ID NO. 4. The humanpolyimmunoglobulin receptor shows extensive sequence homology and has ananalogous domain structure to that of the rabbit polyimmunoglobulinreceptor. See, Kraehenbuhl et al., Trends in Cell Biol., 2:170 (1992).The portions of the human polyimmunoglobulin receptor which areanalogous to the domains and/or amino acid residues sequence of therabbit polyimmunoglobulin receptor are shown in Table 1.

The rat polyimmunoglobulin receptor sequence has been determined andreported by Banting et al., FEBS Lett., 254:177-183 (1989) and EMBL/GeneBank Accession No. X15741. The nucleotide of the rat polyimmunoglobulinreceptor nucleotide sequence is SEQ ID NO. 9 and the corresponding aminoacid residue sequence is SEQ ID NO 10. The rat polyimmunoglobulinreceptor shows extensive sequence homology and has an analogous domainstructure to that of the rabbit and human polyimmunoglobulin receptor.See, Kraehenbuhl et al., T. Cell Biol., 2:170 (1992). The portions ofthe rat polyimmunoglobulin receptor which are analogous to the domainsand/or amino acid residue sequence of the rabbit polyimmunoglobulinreceptor are shown in Table 1.

The bovine polyimmunoglobulin receptor sequence has been determined andreported in EMBL/Gene Bank Accession No. X81371. The bovinepolyimmunoglobulin receptor nucleotide sequence is SEQ ID NO. 5 and thecorresponding amino acid residue sequence is SEQ ID NO. 6. The bovinepolyimmunoglobulin receptor shows extensive sequence homology and has ananalogous domain structure to that of the rabbit and humanpolyimmunoglobulin receptor. The portions of the bovinepolyimmunoglobulin receptor which are analogous to the domains and/oramino acid residues sequence of the rabbit polyimmunoglobulin receptorare shown in Table 1.

The mouse polyimmunoglobulin receptor sequence has been determined andreported by Piskurich et al., J. Immunol., 150:38 (1993) and EMBL/GeneBank U06431. The mouse polyimmunoglobulin receptor nucleotide is SEQ IDNO. 7 and the corresponding amino acid residue sequence is SEQ ID NO. 8.The mouse polyimmunoglobulin receptor shows extensive sequence homologyand has an analogous domain structure to that of the rabbit and humanpolyimmunoglobulin receptor. The portions of the mousepolyimmunoglobulin receptor which are analogous to the domains and/oramino acid residue sequence of the rabbit polyimmunoglobulin receptorare shown in Table 1.

In addition to the above-identified nucleic acid and corresponding aminoacid residue sequences of the polyimmunoglobulin receptor from a varietyof species, the present invention contemplates the use of a portion of apolyimmunoglobulin receptor from any species. The conserved domainstructure of the polyimmunoglobulin receptor between species allows theselection of analogous amino acid residue sequences within eachpolyimmunoglobulin receptor from different species. The presentinvention contemplates the use of such analogous amino acid residuesequences from any polyimmunoglobulin receptor. The analogous sequencesfrom several polyimmunoglobulin receptor amino acid sequences is asshown in Table 1.

TABLE 1 Analogous Regions of the Amino Acid Residue Sequence of ThePolyimmunoglobulin Receptor of Several Species. The nucleotide sequencecoordinates approximately define the boundaries of the domains ofmolecules. Rabbit Bovine Human Rat Mouse (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID

The protection proteins of the present invention may containsubstantially less than the entire amino acid residue sequence of thepolyimmunoglobulin receptor. In preferred embodiments the protectionprotein contains at least a portion of the amino acid residues 1 to 606of the native polyimmunoglobulin receptor of rabbit. Unlike the nativepolyimmunoglobulin receptor, the protection proteins of the presentinvention are derived from precursor proteins that do not contain theentire amino acid residue sequence greater than the amino acid residue627 derived from the native polyimmunoglobulin receptor and thus maycontain more amino acids or fewer amino acids than secretory components.In preferred embodiments, the protection proteins of the presentinvention do not contain the entire amino acid residue sequence greaterthan amino acid residue 606 of the native polyimmunoglobulin receptor ofrabbit. The present invention contemplates using only portions of thenative polyimmunoglobulin receptor sequence as a protection protein. Inother embodiments, it is contemplated that the protection protein mayend at any amino acid between amino acid residue 606 to 627, includingevery amino acid position between 606 and 627, such as 607, 608, 609,610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,624, 625, 626.

In preferred embodiments, a protection protein of the present inventionhas an amino acid sequence which corresponds to one or more of thefollowing amino acid segments:

1) amino acids (AA) corresponding to AA 21-43 of domain I of the rabbitpolyimmunoglobulin receptor;

2) amino acids (AA) corresponding to AA 1-118 of domain I of the rabbitpolyimmunoglobulin receptor;

3) amino acids (AA) corresponding to AA 119-223 of domain II of therabbit polyimmunoglobulin receptor;

4) amino acids (AA) corresponding to AA 224-332 of domain III of therabbit polyimmunoglobulin receptor;

5) amino acids (AA) corresponding to AA 333-441 of domain IV of therabbit polyimmunoglobulin receptor;

6) amino acids (AA) corresponding to AA 442-552 of domain V of therabbit polyimmunoglobulin receptor;

7) amino acids (AA) corresponding to AA of 553 to 606 or 553 to 627 ofdomain VI of the rabbit polyimmunoglobulin receptor; and does notcontain amino acid residues corresponding to AA residues 607 to 755 or628 to 755 of the rabbit polyimmunoglobulin receptor.

It should be noted the exact boundary of a domain may vary withinapproximately 20 amino acids. However, the domain structure andboundaries will be understood by one skilled in the art.

In addition, the present invention contemplates protection proteinending at the following amino acid residues of the rabbitpolyimmunoglobulin receptor or at an amino acid residue whichcorresponds to the following residues but is in the polyimmunoglobulinreceptor of another species: 580-605.

In other preferred embodiments, a protection protein has an amino acidsequence which corresponds to the amino acid sequence of apolyimmunoglobulin receptor for a particular species and which isanalogous to the following amino acid segments:

i) amino acids (AA) corresponding to AA 21-43 of domain I of the rabbitpolyimmunoglobulin receptor;

2) amino acids (AA) corresponding to AA 1-118 of domain I of the rabbitpolyimmunoglobulin receptor;

3) amino acids (AA) corresponding to AA 119-223 of domain II of therabbit polyimmunoglobulin receptor;

4) amino acids (AA) corresponding to AA 224-332 of domain III of therabbit polyimmunoglobulin receptor;

5) amino acids (AA) corresponding to AA 333-441 of domain IV of therabbit polyimmunoglobulin receptor;

6) amino acids (AA) corresponding to AA 442-552 of domain V of therabbit polyimmunoglobulin receptor;

7) amino acids (AA) corresponding to AA of 553-606 or 553-627 of domainVI of the rabbit polyimmunoglobulin receptor; and does not contain aminoacid residues analogous to amino acid residues 607-755 or 630-755 of therabbit polyimmunoglobulin receptor.

In other preferred embodiments, the protection protein comprises domainsI, IV, V and AA 550-606 or 550-627 of domain VI of the rabbitpolyimmunoglobulin receptor or the amino acid sequence from analogousdomains and regions of a polyimmunoglobulin receptor from a differentspecies.

In other embodiments, a protection protein of the present invention hasan amino acid residue sequence which substantially corresponds to atleast a portion of the amino acid residues from the polyimmunoglobulinreceptor of a species which are analogous to amino acid residues 1-627of the rabbit polyimmunoglobulin receptor. This portion of the aminoacid sequence would correspond to at least a portion of theextracellular domains of the receptor of that species.

In preferred embodiments, a protection protein of the present inventionhas an amino acid sequence which substantially corresponds to at least aportion of the amino acid residues from the polyimmunoglobulin receptorof a species which are analogous to amino acid residues 1-606 of therabbit polyimmunoglobulin receptor.

In other preferred embodiments, a protection protein of the presentinvention has an amino acid residue sequence which substantiallycorresponds to or is analogous to (if from a species other than rabbit)at least a portion of the following amino acid residue sequences:

1) amino acids (AA) corresponding to AA 21-43 of domain I of the rabbitpolyimmunoglobulin receptor;

2) amino acids (AA) corresponding to AA 1-118 to of domain I of therabbit polyimmunoglobulin receptor;

3) amino acids (AA) corresponding to AA 119-223 of domain II of therabbit polyimmunoglobulin receptor;

4) amino acids (AA) corresponding to AA 224-332 of domain III of therabbit polyimmunoglobulin receptor;

5) amino acids (AA) corresponding to AA 333-441 of domain IV of therabbit polyimmunoglobulin receptor;

6) amino acids (AA) corresponding to AA 442-552 of domain V of therabbit polyimmunoglobulin receptor;

7) amino acids (AA) corresponding to AA of 553-606 or 553-627 of domainVI of the rabbit polyimmunoglobulin receptor; and does not contain aminoacid residues corresponding to AA 628 to 755 of the rabbitpolyimmunoglobulin receptor.

In other preferred embodiments, the immunoglobulins of the presentinvention have a protection protein which has a first amino acidsequence which substantially corresponds to at least a portion of theamino acid residues 1 to 606 or 1 to 627 of the rabbitpolyimmunoglobulin receptor and has a second amino acid residue sequencecontiguous with said first amino acid sequence, wherein said secondamino acid residue sequence does not have an amino acid residue sequencecorresponding to the transmembrane segment of the rabbitpolyimmunoglobulin receptor.

In more preferred embodiments, the second amino acid residue sequencehas at least a portion of an amino acid sequence which corresponds toamino acid residues 655 to 755 of a polyimmunoglobulin receptor. Inother preferred embodiments, the second amino acid residue is at least aportion of one or more of the following: an intracellular domain of apolyimmunoglobulin molecule, a domain of a member of the immunoglobulingene superfamily, an enzyme, a toxin, or a linker.

The present invention contemplates protection proteins which do not havean amino acid residue corresponding to the transmembrane segment ofrabbit polyimmunoglobulin receptor but may have amino acid residuescorresponding to the intracellular domain of the rabbitpolyimmunoglobulin receptor and this are deletion mutants of thereceptor.

In other embodiments, protection proteins of the present invention havean amino acid sequence which substantially corresponds to at least oneof the extracellular domains of polyimmunoglobulin receptor of aparticular species. The protection protein may have an amino acidsequence of which a segment of that amino acid sequence whichsubstantially corresponds to an extracellular domain of thepolyimmunoglobulin receptor of one species, and a different segment ofthat amino acid sequence may be from a second species and substantiallycorrespond to an extracellular domain from a different species. Thisinvention contemplates embodiments in which a protection protein has anamino acid sequence which has one amino acid sequence segment whichcorresponds to the amino acid sequence of the polyimmunoglobulinreceptor from one species and has a second amino acid sequence withinthe same domain which corresponds to the amino acid and sequence of thepolyimmunoglobulin receptor of a different species.

Thus, the protection protein may have individual domains or portions ofa particular domain that are comprised of amino acid sequences whichcorrespond to the polyimmunoglobulin receptor from different species.

Other embodiments are contemplated in which protection protein hasportions of its amino acid sequence derived from a molecule which is amember of the immunoglobulin superfamily. See, Williams and Barclay,“The Immunoglobulin Superfamily.” In Immunoglobulin Genes, p. 361,Academic Press (Honjo Alt and Rabbits Eds. 1989). These derived portionsmay include amino acid sequences encoding peptides, domains or multipledomains from an immunoglobulin superfamily molecule.

The present invention also contemplates a nucleotide sequence encoding aprotection protein which has a first nucleotide sequence encoding atleast a portion of amino acids 1-606 or 1-627 of the rabbitpolyimmunoglobulin receptor nucleotide sequence and which does not havea nucleotide sequence which encodes a functional transmembrane segment3′ of the first nucleotide sequence. Further preferred embodimentsinclude a second nucleotide sequence located 3′ of the first nucleotidesequence which encodes the amino acids 1-606 or 1-627 of the rabbitpolyimmunoglobulin receptor sequence. This second nucleotide sequencemay encode a variety of molecules including portions of theintracellular domain of rabbit polyimmunoglobulin receptor or anotherpolyimmunoglobulin receptor or a portion of an immunoglobulinsuperfamily molecule. In addition, embodiments are contemplated in whichthis second nucleotide sequence encodes various effector molecules,enzymes, toxins and the like. Preferred embodiments include a secondnucleotide sequence which encodes amino acid residues which correspondto amino acid residues 655 to 775 of the rabbit polyimmunoglobulinreceptor or polyimmunoglobulin receptor from another species.

The present invention also contemplates expression vectors containing anucleotide sequence encoding a protection protein which has beenoperatively linked to for expression. These expression vectors place thenucleotide sequence to be expressed in a particular cell 3′ of apromoter sequence which causes the nucleotide sequence to be transcribedand expressed. The expression vector may also contain various enhancersequences which improve the efficiency of this transcription. Inaddition, such sequences as terminators, polydenylation (poly A) sitesand other 3′ end processing signals may be included to enhance theamount of nucleotide sequence transcribed within a particular cell.

In preferred embodiments, the protection protein is part of animmunoglobulin that is in association with an immunoglobulin derivedheavy chain having at least a portion of an antigen binding domain.Immunoglobulin derived heavy chains containing at least a portion of anantigen binding domain are well known in the art and have beendescribed, for example, by Huse et al., Science, 246:1275 (1989), and byLerner and Sorge, PCT Application WO 90/14430, published Nov. 29, 1990.The disclosure of these documents are hereby incorporated by reference.

In other embodiments, the immunoglobulins of the present inventioncontain a protection protein and immunoglobulin derived heavy chain andimmunoglobulin derived light chain that contain at least a portion of anantigen binding site in association with the immunoglobulin derivedheavy chain. Immunoglobulin light chains having at least a portion of anantigen binding domain are well known in the art and are described inavailable sources. See, for example, Early and Hood, GeneticEngineering, Setlow & Hollaender, (eds.), Vol. 3, Plenum PublishingCorp., New York (1981), pages 157-188; and Kabat et al., Sequences ofImmunologic Interest, National Institutes of Health, Bethesda, Md.(1987). The disclosures of all references cited herein are herebyincorporated by reference.

The immunoglobulin components of the complex (alpha, J, kappa or lambda)can contain all or part of the full length polypeptide. Parts of thesechains may be used to substitute for the whole chain. For instance, theentire immunoglobulin alpha heavy chain may be replaced by the variableregion and only a portion of the alpha constant region sufficient toenable assembly with the other components. Likewise, a truncated kappaor lambda chain, containing only a small section of constant region canreplace the full length kappa or lambda chains. The prerequisite of anycomplex is the ability to bind the protection protein.

In addition to truncated components, the present invention contemplatesthe combination of different types of immunoglobulins. For example, aheavy chain constant region comprising the C_(H)1 and C_(H)2 regions ofIgG followed by the C_(H)2 and C_(H)3 regions derived from an IgA willform a stable complex containing the protection protein. This isspecifically described as an example.

The immunoglobulins containing the protection proteins of the presentinvention preferably contain at least a portion of an IgM or IgA heavychain which allows that immunoglobulin heavy chain to bind toimmunoglobulin J chain and thereby bind to the protection protein. It iscontemplated that the immunoglobulin heavy chain of the presentinvention may be comprised of individual domains selected from the IgAheavy chain or the IgM heavy chain or from some other isotype of heavychain. It is also contemplated that an immunoglobulin domain derivedfrom an immunoglobulin heavy chain other than IgA or IgM may bemolecularly engineered to bind immunoglobulin J chain and thus may beused to produce immunoglobulins of the present invention.

One skilled in the art will understand that immunoglobulins consist ofdomains which are approximately 100-110 amino acid residues. Thesevarious domains are well known in the art and have known boundaries. Theremoval of a single domain and its replacement with a domain of anotherantibody molecule is easily achieved with modern molecular biology. Thedomains are globular structures which are stabilized by intrachaindisulfide bonds. This confers a discrete shape and makes the domains aself-contained unit that can be replaced or interchanged with othersimilarly shaped domains. The heavy chain constant region domains of theimmunoglobulins confer various properties known as antibody effectorfunctions on a particular molecule containing that domain. Exampleeffector functions include complement fixation, placental transfer,binding to staphyloccal protein, binding to streptococcal protein G,binding to mononuclear cells, neutrophils or mast cells and basophils.The association of particular domains and particular immunoglobulinsisotopes with these effector functions is well known and for example,described in Immunology, Roitt et al., Mosby St. Louis, Mo. (1993 3rdEd.)

The immunoglobulins of the present invention may, in addition to theprotection protein, contain immunoglobulin heavy chains, immunoglobulinlight chains, or immunoglobulin J chain bound to the immunoglobulinderived heavy chains. In preferred embodiments, the immunoglobulin ofthe present invention comprises two or four immunoglobulin derived heavychains, together with two or four immunoglobulin light chains and animmunoglobulin J chain bound to at least one of the immunoglobulinderived heavy chains. The immunoglobulin J chain is described and knownin the art. See, for example, M. Koshland, The Immunoglobulin Helper:The J Chain, in Immunoglobulin Genes, Academic Press, London, Pg. 345,(1989) and Matsuuchi et al., Proc. Natl. Acad. Sci. U.S.A., 83:456-460(1986). The sequence of the immunoglobulin J chain is available onvarious data bases in the United States.

The immunoglobulin of the present invention has a protection proteinassociated with at least an immunoglobulin derived heavy chain. Thisassociation may occur by hydrogen bonds, disulfide bonds, covalentbonds, ionic interactions or combinations of these various bonds.Typically, immunoglobulin molecules are held together by disulfide bondsbetween the immunoglobulin heavy chains and immunoglobulin light chains.The interaction of the protection protein with the immunoglobulin is bynon-covalent or disulfide bonding.

The immunoglobulins of the present invention containing the protectionprotein, the immunoglobulin derived heavy chain and optionally animmunoglobulin derived light chain, and J chain are typically bondedtogether by one of the following: hydrogen bonds, disulfide bonds,covalent bonds, ionic interactions or combinations of these bonds. Thepresent invention contemplates molecules in which the required portionsof the immunoglobulin heavy, light and/or J chain have been placed intoa single polypeptide and function to bind antigen and protectionprotein. Examples of such proteins are single-chain antigen-bindingproteins.

The present invention contemplates a method of assembling a multimericimmunoglobulin comprising the steps of: introducing into an organism aDNA segment encoding all or part of an immunoglobulin J chain, and a DNAsegment encoding all or part of an immunoglobulin alpha chain, and a DNAsegment encoding all or part of either an immunoglobulin kappa chain oran immunoglobulin lambda chain; and introducing into the same organism aprotection protein, said protection protein comprising at least asegment of the amino acid residues 1 to residue 606 of the rabbitpolyimmunoglobulin receptor (pIgR) amino acid residue sequence oranalogous amino acid residues from other species such that the segmentis derived from a precursor protein that does not contain the amino acidresidues comprising a functional membrane spanning region nor is thesegment derived from a precursor protein in which the sequence of aminoacid residues from the beginning of the membrane spanning region(approximately residue 630 of rabbit polyimmunoglobulin receptor) to thecarboxyl end of the protein (approximately residue 755 of the rabbitpolyimmunoglobulin receptor) are fully intact. In preferred embodimentsthe precursor protein does not contain amino acid residues greater than606 of the rabbit polyimmunoglobulin receptor or analogous amino acidresidues from other species.

As is understood by those of ordinary skill in the art, a membranespanning region or functional transmembrane segment consists of acontiguous section of amino acid residues containing from about 20 toabout 30 amino acids in which none of the residues is charged, virtuallyall of the residues are hydrophobic or non-polar, and the segment formsan alpha helix. A functional transmembrane segment is capable ofspanning a biomembrane. Membrane spanning regions can be bounded bycharged residues. An example of a membrane spanning region of pIgR isresidues 630 to 653 of the polyimmunoglobulin receptor amino acidresidue sequence of rabbit.

The chains that comprise the immunoglobulin containing the protectionprotein may be derived from precursors containing a signal sequence atthe amino terminal of the protein. Each component can thereby besynthesized into an endomembrane system where assembly occurs. Inaddition to a signal sequence, the various components of the complex mayor may not contain additional signals for N terminal glycosylation orfor various other modifications which can affect the structure of thecomplex. In one embodiment of the invention, the signals forglycosylation (i.e. asparagine-X-serine or threonine or the signals forO-linked glycosylation) are not present or present in more or lessplaces within the nucleotide sequence. The resulting antibody thereforewould contain no carbohydrate, which may be advantageous forapplications in which carbohydrates elicit an immune response.

In preferred embodiments, the immunoglobulin of the present inventioncontains a protection protein associated with an immunoglobulin derivedheavy chain and the protection protein is free from N-linked and/orO-linked oligosaccharides. One skilled in the art will understand that agene coding for a polypeptide having within its amino acid residuesequence the N-linked glycosylation signal asparagine-X-serine/threoninewhere X can be any amino acid residue except possibly proline andaspartic acid, when introduced into a plant cell would be glycosylatedvia oligosaccharides linked to the asparagine residue of the sequence(N-linked). See, Marshall, Ann. Rev. Biochem., 41:673 (1972) andMarshall, Biochem. Soc. Symp., 40:17 (1974) for a general review of thepolypeptide sequences that function as glycosylation signals. Thesesignals are recognized in both mammalian and in plant cells. One skilledin the art will understand that the N-linked glycosylation signal may beeasily removed using common mutagenesis procedures to change the DNAsequence encoding the protection protein of the present invention. Thismutagenesis typically involves the synthesis of oligonucleotide havingthe N-linked glycosylation signal deleted and then preparing a DNAstrand with that oligonucleotide sequence incorporated into it. Suchmutagenesis procedures and reagents are commercially available from manysources such as Stratagene (La Jolla, Calif.).

Assembly of the individual polypeptides that form a multi-peptidemolecule (for example immunoglobulin) may be obtained by expressing in asingle cell by directly introducing all the transgenes encoding theindividual polypeptides into that cell either sequentially or all atonce. The transgenes encoding the polypeptides may be present onindividual constructs or DNA segments or may be contained in a DNAsegment or construct together with one or more other transgenes.

Assembly of these components can be by cross pollination as originallydescribed by Mendel to produce a population of segregants expressing allchains. Previous disclosures have demonstrated this to be an adequatemethod for the assembly and co-segregation of multimericglycoconjugates. The disclosure of U.S. Pat. No. 5,202,422 is herebyincorporated by reference and describes these methods. In a preferredembodiment of the present invention, the antibody molecules contain areduced number of glycans and antibody molecules with no glycans arecontemplated.

The immunoglobulins of the present invention containing the protectionprotein, the immunoglobulin derived heavy chain and optionally animmunoglobulin derived light chain, and J chain may contain a protectionprotein that is free from N-linked oligosaccharides.

The immunoglobulins of the present invention that contain the protectionprotein are preferably therapeutic immunoglobulins that are useful inpreventing a disease in an animal. In preferred embodiments, theimmunoglobulins of the present invention are therapeutic immunoglobulinswhich are capable of binding to mucosal pathogen antigens. In otherpreferred embodiments, the therapeutic immunoglobulins of the presentinvention are capable of preventing dental caries. In the most preferredembodiment, the immunoglobulin of the present invention containing theprotection protein contains an antigen binding domain that is capable ofbinding to an antigen from S. mutans serotypes a, c, d, e, f, g and h(S. mutans c, e and f and S. sobrinus serotypes d and g under newnomenclature). Such antigen binding domains are known in the art andinclude, for example, the binding domains described in U.S. Pat. No.5,352,446, J. K-C. Ma et al., Clin. Exp. Immunol. 77:331 (1989); and J.K-C. Ma et al., Eur. J. Immunol. 24:131-138 (1994); U.S. Pat. No.5,352,446; U.S. Pat. No. 4,594,244; and European Patent Publication 371017 B1. The disclosures of these documents are hereby incorporated byreference. In preferred embodiments, the immunoglobulins of the presentinvention are part of a composition that has a therapeutic activity oneither animals or humans. Examples of therapeutic immunoglobulins arenumerous, however, we envision the most appropriate therapeutic effectto be prophylaxis for mucosal and enteric pathogens by direct oraladministration of the composition derived from an edible plant.

Administration of the therapeutic composition can be before or afterextraction from the plant or other transgenic organism. Once extractedthe immunoglobulins may also be further purified by conventionaltechniques such as size exclusion, ion exchange, or affinitychromatography. In the preferred embodiment, the transgenic organism isan edible plant and administration of the complex is by ingestion afterpartial purification. Plant molecules may be co-administered with thecomplex.

The present invention also contemplates that the relative proportion ofplant-derived molecules and animal-derived molecules can vary.Quantities of specific plant proteins, such as RuBisCo, or chlorophyllmay be as little as 1% of the mass or as much as 99.9% of the mass ofthe extract, excluding water.

The present invention also contemplates the use of the therapeutic plantextract containing immunoglobulins having a protection protein directlywithout any further purification of the specific therapeutic component,e.g. the antibody. Administration may be by topical application, oralingestion or any other method appropriate for delivering the antibody tothe mucosal target pathogen. This form of administration is distinctfrom parenteral applications involving direct injection or comminglingof the therapeutic plant extract with the blood stream.

The present invention also contemplates the use of the therapeutic plantextract containing immunoglobulins having a protection protein aftermanipulating the taste or texture of the extract. Appropriate quantitiesof gelling substances or flavorings could be added to enhance thecontact of the antibody with the target pathogen in, for example, directoral applications.

In preferred embodiments, the immunoglobulins of the present inventionare used to passively immunize an animal against a preselected ligand bycontacting a composition comprising an immunoglobulin containing aprotection protein of the present invention that is capable of binding apreselected ligand with a mucosal surface of an animal. Passiveimmunization requires large amounts of antibody and for wide-spread usethis antibody must be inexpensive.

Immunoglobulin molecules containing protection proteins that are capableof binding a preselected antigen can be efficiently and economicallyproduced in plant cells. In preferred embodiments, the immunoglobulinmolecule is either IgA, IgM, secretory IgM or secretory IgA or animmunoglobulin having a chimeric immunoglobulin heavy or light chain.

The immunoglobulins containing protection proteins are more resistant toproteolysis and denaturation and therefore are desirable for use inharsh environments. Contemplated harsh environments include acidicenvironments, protease containing environments, high temperatureenvironments, and other harsh environments. For example, thegastrointestinal tract of an animal is a harsh environment where bothproteases and acid are present. See, Kobayashi et al., Immunochemistry,10:73 (1973).

Passive immunization of the animal using these more resistantimmunoglobulins of the present invention is produced by contacting theimmunoglobulin containing the protection protein with a mucosal surfaceof the animal. Animals have various mucosal surfaces including thelungs, the digestive tract, the nasopharyngeal cavity, the urogenitalsystem, and the like. Typically, these mucosal surfaces contain cellsthat produce various secretions including saliva, lacrimal fluid, nasalfluid, tracheobronchial fluid, intestinal fluid, bile, cervical fluid,and the like.

In preferred embodiments the immunoglobulins that contain the protectionprotein are immunospecific for a preselected antigen. Typically, thisantigen is present on a pathogen that causes a disease that isassociated with the mucosal surface such as necrotizing enterocolitis,diarrheal disease, ulcers, and cancer caused by carcinogen absorption inthe intestine. See e.g., McNabb and Tomasi, Ann. Revl. Microbiol. 35:477(1981) and Lawrence et al., Science, 243:1462 (1989). Typical pathogensthat cause diseases associated with a mucosal surface include bothbacterial and viral pathogens, such as E. coli., S. typhimurium, V.cholera, H. pylori, and S. mutans. See also, European Patent Application484, 148 A1, published May 6, 1992 and hereby incorporated by reference.The immunoglobulins of the present invention are capable of binding tothese pathogens and preventing them from causing mucosal associateddiseases.

Immunoglobulins capable of binding to S. mutans and preventing dentalcaries have been described in European Patent Specification 371,017which is hereby incorporated by reference. The disclosure of U.S. Pat.No. 5,352,440 is also hereby incorporated by reference.

Therapeutic immunoglobulins of the present invention that containprotection proteins that would be effective against bacterial infectionor carcinomas are contemplated. Monoclonal antibodies with therapeuticactivity have been described in U.S. Pat. Nos. 4,652,448, 4,443,549 and5,183,756 which are hereby incorporated by reference.

In preferred embodiments, the immunoglobulin of the invention are partof a composition which is contacted with the animal mucosal surfacecomprises plant material and an immunoglobulin of the present inventionthat is capable of binding a preselected ligand. The plant materialpresent may be plant cell walls, plant organelles, plant cytoplasms,intact plant cells, viable plants, and the like. This plant cellmaterial is present in a ratio from about 10,000 grams of plant materialto about 100 nanograms of immunoglobulin to about 100 nanograms of plantmaterial for each 10 grams of immunoglobulin present. In more preferredembodiments, the plant material is present in a ratio from about 10,000grams of plant material for each 1 gram of immunoglobulin present toabout a ratio of 100 nanograms of plant material present for each gramof immunoglobulin present. In other preferred embodiments, the plantmaterial is present in a ratio from about 10,000 grams of plant materialfor each milligram of immunoglobulin present to about 1 milligram ofplant material present for each 500 milligram of immunoglobulin present.

In preferred embodiments, the composition containing the immunoglobulinsof the present invention is a therapeutic composition. The preparationof therapeutic compositions which contain polypeptides or proteins asactive ingredients is well understood in the art. Therapeuticcompositions may be liquid solutions or suspensions, solid formssuitable for solution in, or suspension in a liquid prior to ingestionmay also be prepared. The therapeutic may also be emulsified. The activetherapeutic ingredient is typically mixed with inorganic and/or organiccarriers which are pharmaceutically acceptable and compatible with theactive ingredient. The carriers are typically physiologically acceptableexcipients comprising more or less inert substances when added to thetherapeutic composition to confer suitable consistencies and form to thecomposition. Suitable carriers are for example, water, saline, dextrose,glycerol, and the like and combinations thereof. In addition, if desiredthe composition can contain minor amounts of auxiliary substances suchas wetting or emulsifying agents and pH buffering agents which enhancethe effectiveness of the active ingredient. Therapeutic compositorscontaining carriers that have nutritional value are also contemplated.

In embodiments in which a composition containing an immunoglobulinhaving a protection protein of the present invention is applied to thetooth or mouth of a mammal, any convenient method may be used. Methodsfor applying such a composition to the teeth are well known and utilizevarious materials for a variety of purposes. For example, thecomposition may be directly applied to the tooth by painting the surfaceof the tooth with that composition. Alternatively, the composition ofthe present invention may be included in a toothpaste, mouthwash,chewing gum, lozenge or gel that will result in it being applied to theteeth. In some formulations, it may be desirable to provide for aformulation that prolongs the contact of the composition and thereforethe immunoglobulin having the protection protein with the tooth surface.Formulations for this purpose are well known and include suchformulations that may be placed in various dental trays that are used tocover the tooth and other dental apparatuses that are used in adjustingvarious conditions with the teeth.

The exact amount of a composition that must be applied to the teethduring any particular application is not critical because such treatmentmay be easily repeated at a given interval. For example, compositionspresent in toothpaste would be applied to the teeth each time thattoothpaste is used, typically twice per day. For example, the order of10 to 100 micrograms of an immunoglobulin having a protection proteincan be applied to each tooth on each occasion the composition is appliedto the teeth. However, this in no way should be taken as a limitation ona range that may be applied during any particular application asapplications of a composition having more or less immunoglobulin of thepresent invention may be used without detrimental effect. The use ofmuch lower concentrations of an immunoglobulin of the present inventionwould result in, at some point, a reduction in the protection providedby such formulation.

The exact formulation for the composition of the present invention mayvary and will depend on the method of application to be used and thefrequency of that application. In general, it may be any formulationwhich has an appropriate pH and which is free of material which wouldrender the immunoglobulin having the protection protein of the presentinvention ineffective. For example, the compositions of the presentinvention may be applied as a simple aqueous solution in which thecomposition is disbursed at anywhere from 0.1 to 10 milligrams ofimmunoglobulin per 100 microliters of that solution. Generally, such asolution would be applied during dental surgery at a rate ofapproximately 1 to 10 microliters of the solution per tooth.

The formulations of the compositions of the present invention which aredesigned to be self-administered may vary and will be formulated takingin to account the frequency of application of the particular product inwhich is it used.

In preferred embodiments, a composition containing an immunoglobulin ofthe present invention comprises an immunoglobulin molecule that isimmunospecific for a pathogen antigen. Pathogens are any organism thatcauses a disease in another organism. Particularly preferred areimmunoglobulins that are immunospecific for a mucosal pathogen antigen.A mucosal pathogen antigen is present on a pathogen that invades anorganism through mucosal tissue or causes mucosal associated diseases.Mucosal pathogens include lung pathogens, nasal pathogens, intestinalpathogens, oral pathogens, and the like. For a general discussion ofpathogens, including mucosal pathogens, see, Davis et al., Microbiology,3rd ed., Harper and Row, Hagerstown, Md. (1980).

Antibodies immunospecific for a pathogen may be produced using standardmonoclonal antibody production techniques. See, Antibodies: A LaboratoryManual, Harlow et al., eds., Cold Spring Harbor, N.Y. (1988). The genescoding for the light chain and heavy chain variable regions can then beisolated using the polymerase chain reaction and appropriately selectedprimers. See, Orlandi et al., Proc. Natl. Acad. Sci., U.S.A., 86:3833(1989) and Huse et al., Science, 246:1275 (1989). The variable regionsare then inserted into plant expression vectors, such as the expressionvectors described by Hiatt et al., Nature, 342:76-78 (1989).

In a preferred embodiment, the immunoglobulin of the present inventionis immunospecific for an intestinal pathogen antigen. Particularlypreferred are immunoglobulins immunospecific for intestinal pathogenssuch as bacteria, viruses, and parasites that cause disease in thegastrointestinal tract, such as E. coli, Salmonellae, Vibrio cholerae,Salmonellae typhimurium, Shigella and H. pylori.

In other preferred embodiments, the immunoglobulin containing theprotection protein present in the composition is an immunoglobulinmolecule that is immunospecific for a dental pathogen such asStreptococcus mutans and the like. Particularly preferred areimmunoglobulins immunospecific for a Streptococcus mutans antigen suchas the immunoglobulin produced by hybridoma 15B2 (ATCC No. HB 8510); thehybridoma deposited as European Collection of Animal cells Deposit No.86031901; and the Guy's 13 monoclonal antibody described by Ma et al.,Eur. J. Immunol., 24:131 (1994) and Smith and Lehner, Oral Micro.Immunol., 4:153 (1989).

The present invention contemplates producing passive immunity in ananimal, such as vertebrate. In preferred embodiments, passive immunityis produced in fish, birds, reptiles, amphibians, or insects. In otherpreferred embodiments passive is produced in an mammal, such as a human,a domestic animal, such as a ruminant, a cow, a pig, a horse, a dog, acat, and the like. In particularly preferred embodiments, passiveimmunity is produced in an adult or child mammal.

In preferred embodiments, passive immunity is produced in an animal,such as a mammal that is weaned and therefore no longer nurses to obtainmilk from its mother. Passive immunity is produced in such an animal byadministering to the animal a sufficient amount of compositioncontaining an immunoglobulin containing a protection proteinimmunospecific for a preselected ligand to produce a prophylacticconcentration of the immunoglobulin within the animal. A prophylacticconcentration of an immunoglobulin is an amount sufficient to bind to apathogen present and prevent that pathogen from causing detectabledisease within the animal. The amount of composition containing theimmunoglobulin of the present invention required to produce aprophylactic concentrations will vary as is well known in the art withthe size of the animal, the amount of pathogen present, the affinity ofthe particular immunoglobulin for the pathogen, the efficiency withwhich the particular immunoglobulin is delivered to its active locationwithin the animal, and the like.

C. Eukaryotic Cells Containing Immunoglobulins Having a ProtectionProtein

The present invention contemplates eukaryotic cells, including plantcells, containing immunoglobulins of the present invention. The presentinvention also contemplates plant cells that contain nucleotidesequences encoding the various components of the immunoglobulins of thepresent invention. One skilled in the art will understand that thenucleotide sequences that encode the protection protein and the variousimmunoglobulin heavy and light chains and J chain will typically beoperably linked to a promoter and present as part of an expressionvector or cassette.

After the immunoglobulin heavy and light chain genes, and J chain genesare isolated, they are typically operatively linked to a transcriptionalpromoter in an expression vector.

Expression of the components in the organism of choice can be derivedfrom an independently replicating plasmid, or from a permanent componentof the chromosome, or from any piece of DNA which may transiently giverise to transcripts encoding the components. Organisms suitable fortransformation can be either prokaryotic or eukaryotic. Introduction ofthe components of the complex can be by direct DNA transformation, byballistic delivery into the organism, or mediated by another organism asfor example by the action of recombinant Agrobacteria on plant cells.Expression of proteins in transgenic organisms usually requiresco-introduction of an appropriate promoter element and polyadenylationsignal. In one embodiment of the invention, the promoter elementpotentially results in the constitutive expression of the components inall of the cells of a plant. Constitutive expression occurring in mostor all of the cells will ensure that precursors can occupy the samecellular endomembrane system as might be required for assembly to occur.

Expression vectors compatible with the host cells, preferably thosecompatible with plant cells are used to express the genes of the presentinvention. Typical expression vectors useful for expression of genes inplants are well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. in Enzymol., 153:253-277 (1987). However, severalother expression vector systems are known to function in plants. See forexample, Verma et al., PCT Publication No. WO87/00551; and Cocking andDavey, Science, 236:1259-1262 (1987).

The expression vectors described above contain expression controlelements including the promoter. The genes to be expressed areoperatively linked to the expression vector to allow the promotersequence to direct RNA polymerase binding and synthesis of the desiredpolypeptide coding gene. Useful in expressing the genes are promoterswhich are inducible, viral, synthetic, constitutive, and regulated. Thechoice of which expression vector and ultimately to which promoter anucleotide sequence encoding part of the immunoglobulin of the presentinvention is operatively linked depends directly, as is well known inthe art, on the functional properties desired, e.g. the location andtiming of protein expression, and the host cell to be transformed, thesebeing limitations inherent in the art of constructing recombinant DNAmolecules. However, an expression vector useful in practicing thepresent invention is at least capable of directing the replication, andpreferably also the expression of the polypeptide coding gene includedin the DNA segment to which it is operatively linked.

In preferred embodiments, the expression vector used to express thegenes includes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. A preferred drugresistance marker is the gene whose expression results in kanamycinresistance, i.e., the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II and nopaline synthase 3′nontranslated region described by Rogers et al., in Methods For PlantMolecular Biology, a Weissbach and H. Weissbach, eds., Academic PressInc., San Diego, Calif. (1988). A useful plant expression vector iscommercially available from Pharmacia, Piscataway, N.J.

Expression vectors and promoters for expressing foreign proteins inplants have been described in U.S. Pat. Nos. 5,188,642; 5,349,124;5,352,605, and 5,034,322 which are hereby incorporated by reference.

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracks can be added to the DNA segment to be inserted and tothe vector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Alternatively, synthetic linkers containing one or more restrictionendonuclease sites can be used to join the DNA segment to the expressionvector. The synthetic linkers are attached to blunt-ended DNA segmentsby incubating the blunt-ended DNA segments with a large excess ofsynthetic linker molecules in the presence of an enzyme that is able tocatalyze the ligation of blunt-ended DNA molecules, such as bacteriaphage T4 DNA ligase. Thus, the products of the reaction are DNA segmentscarrying synthetic linker sequences at their ends. These DNA segmentsare then cleaved with the appropriate restriction endonuclease andligated into an expression vector that has been cleaved with an enzymethat produces termini compatible with those of the synthetic linker.Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources including NewEngland BioLabs, Beverly, Mass.

The nucleotide sequences encoding the protection protein and any otherof the immunoglobulins of the present invention are introduced into thesame plant cell either directly or by introducing each of the componentsinto a plant cell and regenerating a plant and cross-hybridizing thevarious components to produce the final plant cell containing all therequired components.

Any method may be used to introduce the nucleotide sequences encodingthe components of the immunoglobulins of the present invention into aeukaryotic cell. For example, methods for introducing genes into plantsinclude Agrobacterium-mediated plant transformation, protoplasttransformation, gene transfer into pollen, injection into reproductiveorgans and injection into immature embryos. Each of these methods hasdistinct advantages and disadvantages. Thus, one particular method ofintroducing genes into a particular eukaryotic cell or plant species maynot necessarily be the most effective for another eukaryotic cell orplant species.

Agrobacterium tumefaciens-mediated transfer is a widely applicablesystem for introducing genes into plant cells because the DNA can beintroduced into whole plant tissues, bypassing the need for regenerationof an intact plant from a protoplast. The use of Agrobacterium-mediatedexpression vectors to introduce DNA into plant cells is well known inthe art. See, for example, the methods described by Fraley et al.,Biotechnology, 3:629 (1985) and Rogers et al., Methods in Enzymology,153:253-277 (1987). Further, the integration of the Ti-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences andintervening DNA is usually inserted into the plant genome as describedby Spielmann et al., Mol. Gen. Genet., 205:34 (1986) and Jorgensen etal., Mol. Gen. Genet., 207:471 (1987). Modern Agrobacteriumtransformation vectors are capable of replication in Escherichia coli aswell as Agrobacterium, allowing for convenient manipulations asdescribed by Klee et al., in Plant DNA Infectious Agents, T. Hohn and J.Schell, eds., Springer-Verlag, New York (1985) pp. 179-203. Furtherrecent technological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described by Rogers etal., Methods in Enzymology, 153:253 (1987), have convenient multi-linkerregions flanked by a promoter and a polyadenylation site for directexpression of inserted polypeptide coding genes and are suitable forpresent purposes.

Agrobacterium-mediated transformation of leaf disks and other tissuesappears to be limited to plant species that Agrobacterium tumefaciensnaturally infects. Thus, Agrobacterium-mediated transformation is mostefficient in dicotyledonous plants. However, the transformation ofAsparagus using Agrobacterium can also be achieved. See, for example,Bytebier, et al., Proc. Natl. Acad. Sci., 84:5345 (1987).

In those plant species where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene transfer. However, few monocots appear to be naturalhosts for Agrobacterium, although transgenic plants have been producedin asparagus using Agrobacterium vectors as described by Bytebier etal., Proc. Natl. Acad. Sci. U.S.A., 84:5345 (1987). Therefore,commercially important cereal grains such as rice, corn, and wheat mustbe transformed using alternative methods. Transformation of plantprotoplasts can be achieved using methods based on calcium phosphateprecipitation, polyethylene glycol treatment, electroporation, andcombinations of these treatments. See, for example, Potrykus et al.,Mol. Gen. Genet., 199:183 (1985); Lorz et al., Mol. Gen. Genet., 199:178(1985); Fromm et al., Nature, 319:791 (1986); Uchimiya et al., Mol. Gen.Genet., 204:204 (1986); Callis et al., Genes and Development, 1:1183(1987); and Marcotte et al., Nature, 335:454 (1988).

Application of these systems to different plant species depends upon theability to regenerate that particular plant species from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described in Fujimura et al., Plant Tissue Culture Letters, 2:74(1985); Toriyama et al., Theor Appl. Genet., 73:16 (1986); Yamada etal., Plant Cell Rep., 4:85 (1986); Abdullah et al., Biotechnology,4:1087 (1986).

To transform plant species that cannot be successfully regenerated fromprotoplast, other ways to introduce DNA into intact cells or tissues canbe utilized. For example, regeneration of cereals from immature embryosor explants can be effected as described by Vasil, Biotechnology, 6:397(1988). In addition, “particle gun” or high-velocity microprojectiletechnology can be utilized as well. Using such technology, DNA iscarried through the cell wall and into the cytoplasm on the surface ofsmall (0.525 um) metal particles that have been accelerated to speeds ofone to several hundred meters per second as described in Klein et al.,Nature, 327:70 (1987); Klein et al., Proc. Natl. Acad. Sci. U.S.A.,85:8502 (1988); and McCabe et al., Biotechnology, 6:923 (1988). Themetal particles penetrate through several layers of cells and thus allowthe transformation of cells within tissue explants. Metal particles havebeen used to successfully transform corn cells and to produce fertile,stably transformed tobacco and soybean plants. Transformation of tissueexplants eliminates the need for passage through a protoplast stage andthus speeds the production of transgenic plants.

DNA can be introduced into plants also by direct DNA transfer intopollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlantMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codinggenes can be obtained by injection of the DNA into reproductive organsof a plant as described by Pena et al., Nature, 325:274 (1987). DNA canalso be injected directly into the cells of immature embryos and therehydration of desiccated embryos as described by Neuhaus et al., Theor.Apl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986).

The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, A. Weissbach and H. Weissbach, eds., AcademicPress, Inc., San Diego, Calif. (1988). This regeneration and growthprocess includes the steps of selection of transformant cells andshoots, rooting the transformant shoots and growth of the plantlets insoil.

The regeneration of plants containing the foreign gene introduced byAgrobacterium tumefaciens from leaf explants can be achieved asdescribed by Horsch et al., Science, 227:1229-1231 (1985). In thisprocedure, transformants are grown in the presence of a selection agentand in a medium that induces the regeneration of shoots in the plantspecies being transformed as described by Fraley et al., Proc. Natl.Acad. Sci. U.S.A., 80:4803 (1983). This procedure typically producesshoots within two to four weeks and these transformant shoots are thentransferred to an appropriate root-inducing medium containing theselective agent and an antibiotic to prevent bacterial growth.Transformant shoots that rooted in the presence of the selective agentto form plantlets are then transplanted to soil to allow the productionof roots. These procedures will vary depending upon the particular plantspecies employed, such variations being well known in the art.

The immunoglobulins of the present invention may be produced in anyplant cell including plant cells derived from plants that aredicotyledonous or monocotyledonous, solanaceous, alfalfa, legumes, ortobacco.

Transgenic plants of the present invention can be produced from anysexually crossable plant species that can be transformed using anymethod known to those skilled in the art. Useful plant species aredicotyledons including tobacco, tomato, the legumes, alfalfa, oaks, andmaples; monocotyledons including grasses, corn, grains, oats, wheat, andbarley; and lower plants including gymnosperms, conifers, horsetails,club mosses, liver warts, horn warts, mosses, algaes, gametophytes,sporophytes of pteridophytes.

The plant cells of the present invention may in addition to theprotection protein and the immunoglobulin derived heavy chain alsocontains a nucleotide sequence encoding an immunoglobulin derived lightchain having at least a portion of an antigen binding domain.

The plant cells of the present invention may have an antigen bindingdomain that is capable of binding an antigen from S. mutans serotypes a,c, d, e, f, g, and h (S. mutans serotypes c, e, and f; and S. sobrinusserotypes d and g under new nomenclature) on the immunoglobulin derivedheavy and light chains. The antigen binding domain present in theseplant cells also can be able to bind to the responsible mucosalpathogens and prevent dental caries.

The plant cells of the present invention may be part of a plant and makeup one of the following types of plants: dicotyledonous,monocotyledonous, solanaceous, alfalfa, tobacco or other type of plant.

D. Compositions Containing Immunoglobulins Having Protection Proteins

The present invention contemplates compositions of matter that compriseimmunoglobulins of the present invention and plant macromolecules.Typically these plant macromolecules are derived from any plant usefulin the present invention. The plant macromolecules are present togetherwith an immunoglobulin of the present invention for example, in a plantcell, in an extract of a plant cell, or in a plant. Typical plantmacromolecules associated with the immunoglobulins of the presentinvention in a composition are ribulose bisphosphate carboxylase, lightharvesting complex, (LH6) pigments, secondary metabolites orchlorophyll. The compositions of the present invention have animmunoglobulin of the present invention present in a concentration ofbetween 1% and 99% mass excluding water. Other preferred compositionsinclude compositions having the immunoglobulins of the present inventionpresent at a concentration of between 1% and 50% mass excluding water.Other preferred compositions include immunoglobulins at a concentrationof 1% to 25% mass excluding water.

The compositions of the present invention contain plant macromoleculesat a concentration of between 1% and 99% mass excluding water. Typicallythe mass present in the composition will consist of plant macromoleculesand immunoglobulins of the present invention. When the immunoglobulinsof the present invention are present at a higher or lower concentrationthe concentration of plant macromolecules present in the compositionwill vary inversely. In preferred embodiments the composition of plantmacromolecules are present in a concentration of between 50% and 99%mass excluding water. In the most preferred compositions, the plantmacromolecules are present in a concentration of between 75% and 99%mass excluding water.

The present invention contemplates a composition of matter comprisingall or part of the following: an IgA heavy chain, a kappa or lambdachain, a J chain. These components form a complex and are attached tothe protection protein as defined earlier. The composition also containsmolecules derived from a plant. This composition may also be obtainedafter an extraction process yielding functional antibody andplant-derived molecules.

The extraction method comprises the steps of applying a force to a plantcontaining the complex whereby the apoplastic compartment of the plantis ruptured releasing said complex. The force involves shear, indyn/cm2, as the primary method of releasing the apoplastic liquid.

The whole plant or plant extract contains an admixture of antibody andvarious other macromolecules of the plant. Among the macromoleculescontained in the admixture is ribulose bisphosphate carboxylase(RuBisCo) or fragments of RuBisCo. Another macromolecule is LHCP.Another molecule is chlorophyll.

Shear force is a useful component of the overall force applied to theplant for disruption of apoplastic spaces. Other types of force may alsobe included to optimize the effects of shear. Direct pressure, forexample, measured in lbs/in2, may enhance the effects of the apparatusused to apply shear. Commonly used homogenization techniques which arenot appropriate for antibody extraction involve the use of high speedblades or cylinders which explosively destroy all plant structures.

The compositions of the present invention may contain an immunoglobulinof the present invention and plant molecules that are derived from adicotyledonous, monocotyledonous, solanaceous, alfalfa, tobacco or otherplant. The plant molecules present in the compositions of the presentinvention can be ribulose bisphosphate carboxylase, light harvestingcomplex, pigments, secondary metabolites, chlorophyll or other plantmolecules.

Other useful methods for preparing composition containingimmunoglobulins having protection protein include extraction withvarious solvents and application of vacuum to the plant material. Thecompositions of the present invention may contain immunoglobulins of thepresent in a concentration of between 1% and 99% mass excluding water.The compositions of the present invention may contain plantmacromolecules in a concentration of between 1% and 99% mass excludingwater.

Therapeutic compositions containing immunoglobulins of the presentinvention and plant macromolecules may be produced by processing a plantof the present invention by shearing under pressure a portion of thatplant to produce a pulp containing the therapeutic immunoglobulin andplant macromolecules in a liquid derived from the apoplast or symplastof the plant which also contains the solid plant derived material.Further processing may be accomplished by separating the solid plantderived material from the plant derived liquid containing theimmunoglobulins of the present invention. The starting material for sucha process may include plant leaves, stem, roots, tubers, seeds, fruit orthe entire plant. Typically, this processing is accomplished by amechanical device which releases liquid from the apoplast or symplast ofthe plant. Additional processing steps may include separation of thesolid plant derived material from the liquid using centrificationsettling flocculation or filtration. One skilled in the art willunderstand that these separation methods result in removing the solidplant derived material from the liquid including the immunoglobulins ofthe present invention. The methods of the present invention may produceimmunoglobulins containing a protection protein and an immunoglobulinderived heavy chain that is comprised of domains or portions ofimmunoglobulin alpha chain and immunoglobulin gamma chain. The methodsof the present invention may produce immunoglobulins containing aprotection protein and an immunoglobulin derived light chain that iscomprised of domains or portions of immunoglobulin kappa or lambdachain.

The methods of the present invention are operable on plant cells or partof a plant. The methods of the present invention may also includedmethods that further comprise growing the plant. The methods of thepresent invention may be applied to any plant including dicotyledonous,monocotyledonous, solanaceous, leguminous, alfalfa or tobacco plant. Themethods of the present invention may be used to extract immunoglobulinsfrom a portion of the plant such as a leaf, stem, root, tuber, seeds,fruit or entire plant. The methods of the present invention may use amechanical device to shear the plants to release liquid from theapoplast or symplast of the plant. The plant pulp of the presentinvention may be separated to remove the solid plant material using oneof the following methods: centrifugation, settling, flocculation orfiltration.

E. Methods of Producing Immunoglobulins Containing Protection Proteins

The present invention contemplates methods of producing animmunoglobulin containing a protection protein comprising the steps of:

-   -   (a) Introducing into the plant cell an expression vector        containing a nucleotide sequence encoding a protection protein        operatively linked to a transcriptional promoter; and    -   (b) Introducing into the same plant cell an expression vector        containing a nucleotide sequence encoding an immunoglobulin        derived heavy chain having at least a portion of an antigen        binding domain operatively linked to a transcriptional promoter.

The methods of the present invention optionally include introducing intothe plant cell containing the expression vector with the nucleotidesequences for the protection protein and the immunoglobulin derivedheavy chain a nucleotide sequence encoding an immunoglobulin derivedlight chain at least having a portion of an antigen binding domainoperatively linked to a transcriptional promoter. Methods are alsocontemplated that introduce into a cell that already contains nucleotidesequences and promoters operatively linked to encode a protectionprotein and an immunoglobulin heavy chain and an immunoglobulin lightchain, a promoter operatively linked to a nucleotide sequence encoding Jchain. This results in a cell containing the nucleotide sequencesoperatively linked to promoters for an immunoglobulin heavy chain and animmunoglobulin light chain, J chain and a protection protein.

The plant cells of the present invention may be present as part of aplant that is capable of growth. Particularly useful plants for thisinvention include dicotyledonous, monocotyledonous, solanaceous,legumes, alfalfa, tomato, and tobacco plants.

The methods of the present invention include producing an assembledimmunoglobulin having heavy, light and J chains and a protection proteinwithin a eukaryotic cell. This eukaryotic cell is produced byintroducing into that cell nucleotide sequences operatively linked forexpression encoding an immunoglobulin derived heavy chain having atleast a portion of an antigen binding domain, an immunoglobulin derivedlight chain having at least a portion of an antigen binding domain, animmunoglobulin J chain, and a protection protein. These nucleotidesequences are operatively linked for expression by attaching appropriatepromoters to each individual nucleotide sequence or to more than onenucleotide sequence thereby placing two nucleotide sequences encodingvarious molecules in tandem.

The eukaryotic cell produced by the present methods which contains thesenucleotide sequences encoding the immunoglobulin heavy, light and Jchains and the protection protein is maintained under conditions whichallow those molecules to reproduce and assemble into an immunoglobulinwhich contains the protection proteins of the present invention.

The present invention also contemplates methods for making a particularimmunoglobulin or antigen binding domain or domains of an immunoglobulinresistant to environmental conditions and more stable by operativelylinking a nucleotide sequence encoding at least a portion of an antigenbinding domain derived from an immunoglobulin heavy chain to anucleotide sequence encoding at least one domain derived from animmunoglobulin α or μ heavy chain to form a nucleotide sequence encodinga chimeric immunoglobulin heavy chain. That nucleotide sequence encodingthe chimeric immunoglobulin heavy chain is expressed in a eukaryoticcell which also contains at least one other molecule such as aprotection protein, an immunoglobulin derived light chain having atleast a portion of an antigen binding domain and an immunoglobulin Jchain. In preferred embodiments, the cell contains all of the moleculesincluding an immunoglobulin derived light chain having an antigenbinding domain which is complementary to the antigen binding domainpresent on the immunoglobulin derived heavy chain. This method allowsthe chimeric immunoglobulin heavy chain to assemble with at least oneother molecule, for example, the immunoglobulin derived light chainhaving the complementary antigen binding domain and an immunoglobulin Jchain and the protection protein to form an immunoglobulin containingthe protection protein which is resistant to environmental conditions.

These immunoglobulins are resistant to environmental conditions and thusmore stable when subjected to elevated or reduced temperatures, high orlow pH, high ionic or low ionic concentrations proteolytic enzymes andother harsh conditions. Such harsh conditions are typically found in theenvironment within natural water sources, within the human body, forexample within the gut and on mucosal surfaces, and on the surface of ananimal such as a mammal.

F. Chimeric Immunoglobulins Containing Protection Proteins

The present invention contemplates immunoglobulins containing aprotection protein in which the immunoglobulin domains comprising theheavy and light chain are derived from different isotopes of eitherheavy or light chain immunoglobulins. One skilled in the art willunderstand that using molecular techniques these domains can besubstituted for a similar domain and thus produce an immunoglobulin thatis a hybrid between two different immunoglobulin molecules. Thesechimeric immunoglobulins allow immunoglobulins containing protectionproteins to be constructed that contain a variety of different anddesirable properties that are conferred by different immunoglobulindomains.

The present invention also contemplates chimeric immunoglobulins,including heavy, light and J chain which contain less than an entiredomain derived from a different molecule. The same molecular techniquesmay be employed to produce such chimeric immunoglobulins.

In preferred embodiments, the immunoglobulins of the present inventioncontain at least the C_(H)1, C_(H)2, C_(H)3, domain of mouse IgG, IgG1,IgG2A, IgG2B, IgG3, IgA, IgE, or IgD. Other preferred embodiments of thepresent invention contain immunoglobulin domains that include at leastthe Cμ1, Cμ2, Cμ3, or Cμ4 domain of mouse IGM. Preferred immunoglobulinsinclude immunoglobulins that contain the domains of Cε2, Cε3, and Cε4 ofmouse immunoglobulin IGE.

The present invention also contemplates chimeric immunoglobulins derivedfrom human immunoglobulins. These chimeric immunoglobulins containdomains from two different isotopes of human immunoglobulin. Preferredimmunoglobulins include immunoglobulins that contain immunoglobulindomains including at least the C_(H)1, C_(H)2, or C_(H)3 of human IgG,IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgD. Other preferredimmunoglobulins include immunoglobulins that contain domains from atleast the C_(H)1, C_(H)2, C_(H)3, or C_(H)4 domain of human IgM or IgEThe present invention also contemplates immunoglobulins that containimmunoglobulin domains derived from at least two different isotopes ofmammalian immunoglobulins. Generally, any of the mammalianimmunoglobulins can be used in the preferred embodiments, such as thefollowing isotopes: any isotype of IgG, any isotype of IgA, IgE, IgD orIgM. The immunoglobulins of the present invention contained at least oneof the constant region domains from two different isotopes of mammalianimmunoglobulin.

The present invention also contemplates immunoglobulins that containimmunoglobulin domains derived from two different isotopes of rodentimmunoglobulin. The isotopes of rodent immunoglobulin are well known inthe art. The immunoglobulins of the present invention may containimmunoglobulin derived heavy chains that include at least one of thefollowing immunoglobulin domains: the C_(H)1, C_(H)2, or C_(H)3 domainof a mouse IgG, IgG1, IgG2a, IgG2b, IgG3, IgA, IgE, or IgD; the C_(H)1,C_(H)2, C_(H)3, C_(H)4 domain of mouse IgE or IgM; the C_(H)1, C_(H)2,or C_(H)3 domain of a human IgG, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, orIgD; the C_(H)1, C_(H)2, C_(H)3, C_(H)4 domain of human IgM or IgE; theC_(H)1, C_(H)2, or C_(H)3 domain of an isotype of mammalian IgG, anisotype of IgA, IgE, or IgD; the C_(H)1, C_(H)2, C_(H)3, C_(H)4 domainof a mammalian IgE or IgM; the C_(H)1, C_(H)2, or C_(H)3 domain of anisotype of rodent IgG, IgA, IgE, or IgD; the C_(H)1, C_(H)2, C_(H)3,C_(H)4 domain of a rodent IgE or IgM; the C_(H)1, C_(H)2, or C_(H)3domain of an isotype of animal IgG, an isotype of IgA, IgE, or IgD; andthe C_(H)1, C_(H)2, C_(H)3, C_(H)4 domain of an animal IgE or IgM. Thepresent invention also contemplates the replacement or addition ofprotein domains derived from molecules that are members of theimmunoglobulin superfamily. The molecules that belong to theimmunoglobulin superfamily have amino acid residue sequence and nucleicacid sequence homology to immunoglobulins. The molecules that are partof the immunoglobulin superfamily can be identified by amino acid ornucleic acid sequence homology. See, for example, p. 361 ofImmunoglobulin Genes, Academic Press (1989).

Tetratransgenic Organisms:

The present invention also contemplates a tetratransgenic organism whichis comprised of cells having incorporated into the nucleic acid of thatcell or plant within the cell four different transgenes, each encoding adifferent polypeptide. These transgenes are different in that themessenger RNA and polypeptides produced from that transgene aredifferent from the messenger RNA and polypeptides produced from theother of the four transgenes. Thus, the number of transgenes referred toin the present invention does not include multiple copies of the sametransgene as is commonly found in transgenic organisms. The presentinvention is directed to transgenic organisms having four transgeneswhich are not identical copies of other transgenes. The presentinvention does not exclude the possibility that each of the fourdifferent transgenes may be present in multiple copies. However, atleast four separate transgenes that are different are present within thecells of the transgenic organism.

In addition, the present invention contemplates that four differenttransgenes are related in that the transgenes encode a polypeptide thatis part of a multipolypeptide molecule. Therefore, the present inventioncontemplates that each individual polypeptide chain of a multipeptidemolecule would be present on a transgene within a cell of the transgenicorganism. The expression of each individual different polypeptide of themultipeptide molecule allows the different polypeptides to associatetogether to form the multipeptide molecule within the transgenicanimal's cells. Thus, the present invention does not include within thefour different transgenes in each individual cell, transgenes whichencode polypeptides which do not associate together to perform amultipeptide molecule. Examples of such transgenes encoding moleculesthat do not associate together are polypeptides for antibioticresistance such as kanamycin or neomycin or thymidine kinase.

In preferred embodiments, the transgenes present within a transgenicorganism of the present invention encode the following four differentpolypeptides: a protection protein; an immunoglobulin derived heavychain having at least a portion of an antigen binding domain; animmunoglobulin derived light chain having at least a portion of anantigen binding domain; and an immunoglobulin J chain. In otherpreferred embodiments, one of the transgenes present in the transgenicorganism encodes a chimeric immunoglobulin heavy, light or J chain. Inother preferred embodiments, a transgene of the transgenic organisms ofthe present invention encode either an immunoglobulin heavy chainderived at least in part from an IgA or a IgM immunoglobulin. Otherpreferred embodiments include transgenic organisms containing transgeneswhich encode at least a portion of the amino acid sequence derived froman immunoglobulin heavy chain derived from either an IgA or IgMimmunoglobulin heavy chain.

The present invention contemplates transgenic organisms includingmammals, plants, rodents, reptiles, insects, amphibians, fishes or otherorganisms. In preferred embodiments, the transgenic organism of thepresent invention is a plant or a mammal. Methods of producing suchorganisms are well known. See, i.e., U.S. Pat. Nos. 4,736,866;4,607,388; 4,870,009 and 4,873,191 which are hereby incorporated byreference.

The present invention also contemplates immunoglobulin that containimmunoglobulin derived heavy or immunoglobulin derived light chains thatcontain immunoglobulin domains which have been engineered to make thosedomains less immunogenic in a particular species. Typically, theimmunoglobulin molecule is engineered as to be “humanized” in that itappears to be a human immunoglobulin even though derived from variousother species.

EXAMPLES

The following examples illustrate the disclosed invention. Theseexamples in no way limit the scope of the claimed invention.

1. Construction of DNA Vectors For Expression of Antibodies in Plants.

a. Isolation of the Nucleotide Sequences Encoding the Guy's 13Immunoglobulin

Molecular cloning of the gamma and kappa chains of the Guy's 13 anti-S.mutans antibody was done by the procedures described in Ma et al., Eur.J. Immunol., 24:131 (1994). Briefly, mRNA was extracted from the Guy's13 hybridoma cell line and converted to the cDNA by standard procedures.The cDNA was then amplified with the use of a pair of oligonucleotidesspecifically complementary to either the gamma or kappa cDNA.Amplification was catalyzed by Taq 1 polymerase using a thermal cycleras described. The amplified cDNAs were then digested with theappropriate restriction endonucleases and ligated into the correspondingrestriction site in a standard plant expression vector. Numerousexamples of such vectors have been reported in the literature and aregenerally available. An example of one vector that may be used ispBIN19.

In a related series of experiments, the cDNAs were cloned into thebacterial vector bluescript. Using this construct, the sequence of thegamma and kappa cDNAs was determined using the methods of Maxam andGilbert.

Procedures for cloning antibody cDNAs involving PCR techniques or byconstruction of cDNA libraries followed by ligation of the obtainedcDNAs into appropriate vectors are commonplace techniques which arefamiliar to one of ordinary skill in the art.

b) Hybrid cDNAs Encoding the Guy's 13 Heavy Chain Variable Region, aPart of the Gamma Chain Constant Region and a Part of an Alpha ChainConstant Region.

These constructs were synthesized as described in Ma et al., Eur. J.Immunol., 24:131 (1994) and ligated into the appropriate plantexpression vectors as described above. The final construct had thestructure: Guy's 13 variable region—(IgG1 C_(H)1)-(IgG1 C_(H)2)-(IgAC_(H)2)-(IgA C_(H)3), referred to as IgG2A heavy chain, and Guy's 13variable region—(IgG1CH₁)-(IgACH2)-(IgACH3).

c) The Protection Protein and J Chain.

The cloned rabbit polyimmunoglobulin receptor (pIgR) cDNA was describedby Mostov, Nature, 308:37 (1984) and shown in FIG. 8. The protectionprotein portion was obtained by PCR amplification of a portion of thenucleotide sequence coding for the (pIgR) and ligation into appropriateplant expression vectors as described above. The protection proteinportion of the pIgR used in these constructs included the codon foramino acid number 1 to the codon for amino acid number 606. The methodto accomplish this construction are well known in the art and theoligonucleotides can be selected using the pIgR nucleic acid sequence.

d) cDNAs Encoding Aglycosylated Derivatives of Heavy-Chain ConstantRegions.

Mutagenesis procedures were performed either according to Stratageneprotocols. In each case (i.e. alpha constant region, or protectionprotein) the codon for the asparagine utilized as the attachment sitefor carbohydrates, was changed to a codon for histidine.

2. Production of Transgenic Plants Expressing Therapeutic Antibodies.

Plants and plant cells containing immunoglobulins having a protectionprotein were produced in the following manner.

a) Transfer of Vectors to Agrobacterium tumefaciens

Plant transformation was accomplished by using Agrobacteriumtumefaciens. E. coli DH5α bearing the recombinant pMON530 plantexpression vector were mated with Agrobacterium in the presence of ahelper strain (pRK2013) to provide transfer functions. Alternatively,pMON530 plasmid DNA was introduced into Agrobacteria by directtransformation. In this procedure, the Agrobacterium strain was firstgrown overnight at 28° C. in YEP medium. 2 ml of the overnight culturewas used to inoculate 50 ml of YEP and was grown to an OD₆₀₀ Of 1.0. Thecells were then chilled to 4° C., pelletted by centrifugation andresuspended in 1 ml of ice cold 20 mM CaCl2. About 1 μg of DNA was addedto aliquots of 0.1 ml of ice cold cells. The cells were then rapidlyfrozen by immersion in liquid nitrogen or in a dry ice ethanol bath. Thecells were thawed by incubation at 37° C. for 5 minutes followed by theaddition of 1 ml YEP medium. The cells were allowed to incubate for 2-4hours with gentle shaking. Individual colonies carrying the recombinantvector were isolated by incubation on YEP agar plates containing theappropriate antibiotic.

Agrobacteria containing pMON530 were grown in media containingkanamycin, spectinomycin and chloramphenicol. Small segments of tobaccoleaf were then co-cultivated with the Agrobacterium for 2 days afterwhich the leaf segments were transferred to plates containingcarbenicillin to kill the Agrobacterium. Regeneration of transformedleaf cells into whole plants was allowed to proceed in the presence ofkanamycin selection until the plants were competent for growth in soil.

b) Regeneration of Transformed Tobacco and Petunia Plants.

Leaves from greenhouse grown tobacco or petunia plants were sterilizedin 20% (by volume) Chlorox bleach, 0.1% sodium dodecyl sulfate at roomtemperature for 8 minutes. The leaves were then briefly rinsed in 70%ethanol and allowed to dry in sterile Petri plates.

Leaf discs of approximately 0.5 cm diameter were removed with a sterilehole puncher and placed on agar plates containing MS10 medium (MS10medium per liter: 4.4 g Murashige and Skoog basal salts with minimalorganics [Sigma #M68991, 30 g sucrose, 0.2 mg naphthalene acetic acid, 2mg benzylaminopurine, 0.1 mg nicotinic acid, 0.1 mg pyridoxin, 0.1 mgthiamine, 10 g agar, pH 5.7 with KOH).

A 2 ml aliquot of a suspension of Agrobacterium in LB (approximately1×10⁸ Agrobacteria per ml) was then added to the leaf pieces. Allsurfaces of the leaf discs were contacted with Agrobacteria, excessliquid was poured off the plate, and the discs were co-cultivated withthe bacteria for 2 days at room temperature. The discs were thentransferred to agar plates containing MS10 medium, 50 μg/ml kanamycinand 250 μg/ml carbenicillin (MS10-KC). Regeneration was allowed toproceed with weekly transfer of discs to fresh MS10-KC plates untilregenerating shoots were visible. Shoots were then transferred to agarplates containing MSO-KC medium (MSO-KC per liter: 4.4 g Murashige andSkoog basal salts with minimal organics [Sigma #M68991, 30 g sucrose, 1mg nicotinic acid, 1 mg pyridoxin, 0.1 mg thiamine, 50 μg/ml kanamycinand 250 μg/ml carbenicillin, 10 g agar, pH 5.7 with KOH).

After root formation, plantlets were transferred to soil and grown tomaturity.

c) Regeneration of Transformed Alfalfa Plants.

Alfalfa trifoliates were cut from a greenhouse grown plant andsterilized in 20% (v/v) Chlorox bleach, 0.1% sodium dodecyl sulfate atroom temperature for 8 minutes. The trifoliates were then briefly rinsedin 70% ethanol and allowed to dry in sterile Petri plates.

Leaf pieces of approximately 1 cm×4 mm were cut with a sterile scalpeland placed on agar plates containing B5H medium (B5H medium per liter:3.1 g Gamborg's powdered medium (Sigma #G5893), 500 mg KNO3, 250 mgMgSO4 7H20, 30 g sucrose, 500 mg proline, 1 mg 2,4-dichlorophenoxyaceticacid, 100 μg kinetin, 100 mg inositol, 1 mg nicotinic add, 1 mgpyridoxin, 10 mg thiamine, 10 g agar, 30 ml stock amino acids, pH 5.7with KOH; stock amino acids consist of 26.6 g L-glutamine, 3.32 gserine, 16.8 mg adenine, 333 mg glutathione per liter and are addedafter autoclaving when the medium is approximately 50° C.).

To the leaf pieces was then added 2 ml of a suspension of Agrobacteriumin LB (approximately 1×10⁸ Agrobacteria per ml). All surfaces of theleaf were contacted with Agrobacteria, excess liquid was poured off theplate, and the leaves were co-cultivated with the bacteria for 2 days atroom temperature. The leaf pieces were then transferred to agar platescontaining B5H medium, 25 μg/ml kanamycin and 250 μg/ml carbenicillin(B5H-KC). Regeneration was allowed to proceed with weekly transfer ofleaf pieces to fresh B5H-KC plates until somatic embryos were visible.Embryos were then transferred to agar plates containing BI02Y-KC medium(BI02Y-KC per liter: 25 ml macronutrients, 10 ml micronutrients, 25 mliron, 1 ml vitamins, 1 ml aminos, 2 g yeast extract, 100 mgmyo-inositol, 30 g sucrose, 10 g agar, 25 mg kanamycin, 250 mgcarbenicillin, pH 5.9 with KOH; macronutrients consist of 40 g KNO3, 40g NH4NO3, 13.88 g Ca(NO3)2-4FUO, 1.4 g MgSO4-7H20, 2.6 g KCl, 12 gKh2PO4 per liter yielding a 40× stock; vitamins consist of 100 mgthiamine HCl, 500 mg nicotinic acid, 100 mg pyridoxin-HCl per literyielding a 1000× stock; aminos consists of 2 g per liter glycineyielding a 1000× stock; micronutrients consist of 580 mg MnSO4-4H20,1550 mg ZnSO4-7H20, 160 mg H3BO3, 80 mg KI per liter yielding a 100×stock; iron consists of 1.28 g NaFeEDTA per liter yielding a 40× stock).

After root formation, plantlets were transferred to soil and grown tomaturity.

d) Regeneration of Transformed Tomato Plants.

Cotyledons from 7 day old tomato seedlings were sterilized in 20% (v/v)Chlorox bleach, 0.1% sodium dodecyl sulfate at room temperature for 8minutes. The leaves were then briefly rinsed in 70% ethanol and allowedto dry in sterile Petri plates.

Cotyledon pieces of approximately 0.5 cm diameter were cut with asterile scalpel and placed on agar plates containing MS4 medium (MS4medium per liter: 4.4 g Murashige and Skoog basal salts with minimalorganics [Sigma #M68991, 30 g sucrose, 2 mg zeatin riboside, 5 mgnicotinic acid, 0.5 mg pyridoxin, 0.5 mg thiamine, 1 mM acetosyringone,10 g agar, pH 5.7 with KOH).

To the leaf pieces was then added 2 ml of a suspension of Agrobacteriumin LB (approximately 1×10⁸ Agrobacteria per ml). All surfaces of theleaf discs were contacted with Agrobacteria, excess liquid was pouredoff the plate, and the discs were co-cultivated with the bacteria for 2days at room temperature. The discs were then transferred to agar platescontaining MS4 medium minus acetosyringone containing 50 μg/ml kanamycinand 250 μg/ml carbenicillin (MS4-KC). Regeneration was allowed toproceed with weekly transfer of discs to fresh MS4-KC plates untilregenerating shoots were visible. Shoots were then transferred to agarplates containing MSO-KC medium (MSO-KC per liter: 4.4 g Murashige andSkoog basal salts with minimal organics [Sigma #M68991, 30 g sucrose, 1mg nicotinic acid, 1 mg pyridoxin, 10 mg thiamine, 50 μg/ml kanamycinand 250 μg/ml carbenicillin, 10 g agar, pH 5.7 with KOH).

After root formation, plantlets were transferred to soil and grown tomaturity.

e) Regeneration of Transformed Arabidopsis Plants.

Intact roots derived from Arabidopsis thalliana plants grown in sterileculture were first pretreated on callus inducing medium (CIM) for 3 daysat 28° C. in the dark (CIM medium per liter: 3.1 g Gamborg's powderedmedium (Sigma #G5893), 30 g sucrose, 1 mg 2,4-dichlorophenoxyaceticacid, 100 μg kinetin, 1 mg inositol, 0.1 mg nicotinic acid, 0.1 mgpyridoxin, 0.1 mg thiamine, 8 g agar, pH 5.7 with KOH).

To the intact roots was then added 2 ml of a suspension of Agrobacteriumin LB (approximately 1×10⁸ Agrobacteria per ml). All surfaces of theroots were contacted with Agrobacteria and excess liquid was poured offthe plate. The intact roots were then cut into 5 mm segments and wereco-cultivated with the Agrobacteria for 2 days at 28° C. on CIM plates.The root pieces were then transferred to agar plates containing shootinducing medium (SIM) containing 50 μg/ml kanamycin and 250 μg/mlcarbenicillin (SIM medium per liter: 3.1 g Gamborg's powdered medium(Sigma #G5893), 30 g sucrose, 5 mg N⁶-(2-isopentenyl) adenine, 150 μgindole-3-acetic acid, 1 mg inositol, 0.1 mg nicotinic acid, 0.1 mgpyridoxin, 0.1 mg thiamine, 8 g agar, pH 5.7 with KOH).

Regeneration was allowed to proceed with weekly transfer of root piecesto fresh SIM plates until green regenerating shoots were visible. Shootswere then transferred to agar plates containing EM medium (MSO-KC perliter: 4.4 g Murashige and Skoog basal salts with minimal organics[Sigma #M6899], 10 g sucrose, 1 mg indole-3-butyric acid 1 mg nicotinicacid, 0.1 mg pyridoxin, 0.1 mg thiamine, 250 μg/ml carbenicillin, 8 gagar, pH 5.7 with KOH).

After root formation, plantlets were transferred to soil and grown tomaturity.

3. Identification of Transgenic Plants.

Kanamycin resistant transformants expressing individual immunoglobulinchains were identified by ELISA as described. Further analysis of thetransformants included evaluation of RNA by Northern blotting andevaluation of immunoglobulin polypeptides by Western blotting, both asdescribed in Maniatis et al.

For each immunoglobulin chain, antigenic material, RNA or protein weredetected by the respective assays. Transformants identified as havingthe highest levels of immunoglobulin chains were used in crosspollination protocols.

4. Assembly of Antibodies by Cross Pollination of Transformants.

Cross pollinations were performed in order to obtain plantsco-expressing the various components of the desired antibodies. Thesecrosses yielded alfalfa, tomato, tobacco and Arabidopsis plantscontaining the following assembled components, all of which alsocontained the Guy's 13 antigen binding domain.

Type of Antibody Immunoglobulin Components 1 G1 heavy chain, kappa lightchain 2 G2/A heavy chain, kappa light chain 3 G2/A heavy chain, kappalight chain, J chain 4 G1/A heavy chain, kappa light, J chain,protection protein 5 G1/A heavy chain Kappa light chain

5. Extraction and Evaluation of Guy's 13 Type 1, 2 and 3 & 4 AntibodiesFrom Transgenic Plants.

a) Extraction and Enrichment of Antibody Contained in Leaf.

Leaf pieces were chopped into approximately 1 cm² pieces. The pieceswere then added to a cold solution of TBS having 10 μg/ml leupeptin (1ml TBS per gram of leaf) contained in a chilled porcelain mortar both atapproximately 4° C. Plant liquid was extracted by pulverizing the pieceswith a cold pestle using a circular motion and hand pressure.Pulverizing was continued until the pieces became a nearly uniform pulp(approximately 3 minutes of pulverizing). The pulp was centrifuged at 4°C. and approximately 50,000×g to yield a supernatant devoid of solidplant pieces. Alternatively, the pulp was filtered through a plasticmesh with a pore size of approximately 100 microns.

Depending on the titer of antibody contained in the particular plant,the supernatant was either directly suitable for exposure to antigen orrequired enrichment to a suitable concentration. Yields of IgG1's orIgG/A's in the crude extract were routinely less than 10 μg/ml andaveraged approximately 5 μg/ml. For applications of a Guy's 13 antibodyto mucosal surfaces, enrichment to a concentration of 1 to 4 mg/ml maybe required. As a Type 1, 2 or 3 construct, Guy's 13 antibody required aten to forty-fold enrichment to yield the desired concentration. Thiswas accomplished either by affinity adsorption (utilizing either ProteinA or Protein G), or by lyophilization to remove water. Size exclusionchromatography was also used for enrichment but required completefractionation of the crude extract to yield an antibody of the requiredconcentration. By ELISA assay and by polyacrylamide gel electrophoresis,the co-expressed chains assembled into a complex of approximately180-200 k daltons for types 1 & 2 and approximately 400 k daltons fortype 3. Crude extracts were routinely obtained containing approximatelyof 5-10 μg/ml.

A dramatic increase in antibody accumulation was observed when theprotection protein was crossed into a plant containing Type 3 antibodyyielding a plant containing a Type 4 antibody. By ELISA assay and bypolyacrylamide gel electrophoresis, the co-expressed chains assembledinto a complex of approximately 470,000 daltons. Crude extracts wereroutinely obtained containing in excess of 200 μg/ml with an average ofapproximately 250 μg/ml. Therefore, the SIgA construct of the Guy's 13antibody required minimal enrichment to achieve the targetconcentration. This enrichment could be accomplished by the techniquesdescribed above. Alternatively, it was found that the antibody isreadily separated from the majority of plant molecules by a oneultrafiltration step using membrane with a molecular exclusion of200,000 d.

b) Functionality of the Guy's 13 Type 4 Antibody.

Functional antibody studies were carried out by ELISA. All plantsexpressing antibody light and heavy chains assembled functional antibodythat specifically recognized streptococcal antigen (SA I/II). The levelsof binding and titration curves were similar to those of mouse hybridomacell supernatants. No SA I/II binding was detected with plantsexpressing only J chain or only protection protein. Likewise, wild-typeplants expressing no immunoglobulin showed no detectable levels ofbinding.

In a similar set of experiments, binding of antibody to immobilizedpurified streptococcal antigen or native antigen on the bacterial cellsurface was detected using an anti-secretory component antiserum. Inthese assays, only the Type 4 antibody binding was detected. Thefunctional Type 1, 2 or 3 antibodies did not bind the anti-secretorycomponent antiserum. These results confirm that the protection proteinwas assembled with antibody in the plants expressing Type 4 constructsand in a manner which did not interfere with antigen binding.

6. Expression of Chimeric Immunoglobulins.

The genes encoding the heavy and light chains of a murine monoclonalantibody (mAb Guy's 13) have been cloned and expressed in Nicotianatabacum. Transgenic plants have been regenerated that secretefull-length Guy's 13 antibody. By manipulation of the heavy chain genesequence, constant region domains from an immunoglobulin alpha heavychain have been introduced, and plants secreting Guy's 13 mAb withchimeric gamma/alpha heavy chains have also been produced. For eachplant antibody, light and heavy chains have been detected by Westernblot analysis and the fidelity of assembly confirmed by demonstratingthat the antibody is fully functional, by antigen binding studies.Furthermore, the plant antibodies retained the ability to aggregatestreptococci, which confirms that the bivalent antigen-binding capacityof the full length antibodies is intact.

a) Cloning of Heavy and Light Chain Genes

Messenger RNA was purified from the Guy's 13 and a murine IgA (MOPC315)hybridoma cell line, using an acidguanidiniumthiocyanate-phenol-chloroform extraction. Complementary DNAwas made using Moloney murine leukemia virus reverse transcriptase(Promega, GB). DNA encoding the gamma and kappa chains of Guy's 13 wereamplified by polymerase chain reaction (PCR). The degenerateoligonucleotides used in the PCR were designed to incorporate a 5′terminal XhoI, and a 3′-terminal EcoRI restriction site in the amplifiedDNA fragments. Following restriction enzyme digestion, theimmunoglobulin chain encoding DNA was ligated into a constitutive plantexpression vector (pMON 530), which contains a mouse immunoglobulinleader sequence upstream of the cloning site. The recombinant vector wasused to transform E. coli (DH5-α, Gibco BRL) and screening was bySouthern blotting, using radiolabeled DNA probes derived from theoriginal PCR products. Plasmid DNA was purified from positivetransformants and introduced into Agrobacterium tumefaciens.

A similar approach was used to construct two forms of a hybrid Guy's 13heavy chain. The synthetic oligonucleotides shown in FIG. 1 were used inPCR to amplify the regions: (a) Guy's 13 signal sequence to the 3′ endof Cτ1 domain (J1-J5), (b) Guy's 13 signal sequence to the 3′ end of Cτ2domain (J1-J2), and (c) 5′ end of Cα2 domain to the 3′ terminus of DNAfrom the MOPC 315 hybridoma (J3-J4). The fragments were purified(Geneclean II, Bio 101, La Jolla, Calif.) and digested with HindIII for1 h at 37° C. The Guy's 13 fragments were ligated to the MOPC 315fragment with T4 DNA ligase (Gibco, BRL), at 16° C. for 16 h, and analiquot of the reaction mixture was used as template DNA for a furtherPCR, using the 5′ terminal oligonucleotide for Guy's 13 (J1) and the 3′terminal oligonucleotide for MOPC 315 (J4). Amplified DNA fragments werepurified and ligated into the pMON 530 vector as described above. Thevector used in this procedure did not have a previously inserted mouseleader sequence, as in this case, the DNA encoding the native Guy's 13leader sequence was included in the PCR amplification.

b) Plant Transformation and Regeneration

Leaf discs, about 6 mm in diameter, were cut from surface-sterilizedtobacco leaves (Nicotiana tabacum, var. xanthii) and incubated overnightat 28° C., with a culture of the recombinant A. tumefaciens, containingimmunoglobulin cDNA inserts. The discs were transferred to cultureplates containing a medium that induces regeneration of shoots,supplemented with kanamycin (200 mg/l) and carbenicillin (500 mg/l).Shoots developing after this stage were excised and transplanted onto aroot-inducing medium, supplemented with kanamycin (200 mg/l). Rootedplantlets were transplanted into soil as soon as possible after theappearance of roots. Plants were screened for expression ofimmunoglobulin chains as described below. Those that expressed heavychains were crossed with those expressing light chains, bycross-pollination. The resulting seeds were sown in soil and allowed togerminate. Twenty-two transgenic plants were regenerated fromtransformations with light or heavy chain constructs, as determined byELISA. Crossing of light and heavy chain-secreting plants resulted in3/10 F1 progeny plants expressing kappa and gamma chains together, 4/17plants expressing both kappa and the plant G1/A heavy chain and 3/8plants expressing both kappa and the plant G2/A heavy chain together.

The three different forms of Guy's 13 monoclonal antibody expressed inplants, therefore, all contain the identical light (kappa) chain, butdifferent heavy chains. These will be abbreviated throughout this reportas follows (FIG. 1): Guy's 13 IgG1 with original gamma heavy chain,plant G13, Guy's 13 with IgG/IgA hybrid heavy chain consisting ofvar-τ1-τ2-α2-α3 domains, plant G2/A. The Guy's 13 hybridoma cell culturesupernatant used as a positive control will be abbreviated to Mouse G13.Negative control plants were those that had been transformed with pMON530 vector containing an insert that encodes an irrelevant mouseprotein.

c) Antibody Chain Detection

Production of either gamma, kappa or the gamma/alpha chain hybrids wasdetected by ELISA. Microtiter wells were coated with a goat anti-mouseheavy or light chain-specific IgG (Fisher, USA; Sigma, GB; NordicPharmaceuticals, GB) in 150 mM NaCl, 20 mM Tris-HCl (pH 8) (TBS).Blocking was with 5% non-fat dry milk in TBS at 4° C. overnight. Plantleaves were homogenized in TBS with leupeptin (10 μg/ml) (Calbiochem,USA). The supernatant was added in serial twofold dilutions to themicrotiter plate and incubation was at 4° C. overnight. After washingwith TBS with 0.05% Tween 20, bound immunoglobulin chains were detectedwith the appropriate goat anti-mouse heavy or light chain-specificantibody, conjugated with horseradish peroxidase (Fisher; Sigma; NordicPharmaceuticals), for 2 h at 37° C. Detection was with2,2′-azino-di-(3-ethyl-benzthiazoline-sulfonate) (Boehringer, FRG).

A similar assay was used to determine the concentrations of the murineand plant Guy's 13 antibodies. These were compared with a mouse IgG1 mAb(MOPC 21), and a mouse IgA mAb (TEPC 21) used at known concentrations(Sigma). ELISA plates were coated with an anti-mouse kappa antiserum.After blocking, bound antibody was detected with horseradishperoxidase-labeled anti-mouse gamma or alpha antiserum. Antibodyconcentration was determined by comparison of binding curves for eachantibody.

ELISA was also used to detect the binding function of the assembledantibody. Binding to SA I/II was detected using microtiter plates thathad been coated with purified SA I/II at an optimized concentration of 2μg/ml. The ELISA procedure was as described above. The ability to bindS. mutans or E. coli cells was detected using intact cells (strainsGuy's c, S. mutans and DH5-α, E. coli) that had been grown to stationaryphase, for 18 h at 37° C. and fixed in 10% formalin. All the antibodysolutions were adjusted to an initial concentration of 1.5 μg/ml andused in serial twofold dilutions. Extracts from plants expressing witherGuy's 13 heavy or light chain singly were also included in these assays,to determine if the single immunoglobulin chains exhibited anyantigen-binding activity. Antibodies bound to either cells or purifiedSA I/II were detected using a horseradish peroxidase-conjugated goatanti-mouse light or heavy chain antiserum (Nordic Pharmaceuticals). Theresults are expressed as mean ±standard deviation of duplicate resultsfrom three separate assays.

Competition ELISA was performed on microtiter plates coated withpurified SA I/II as above. The plates were incubated with plant extractsof Guy's 13 hybridoma supernatant at 1.5 μg/ml and serial twofolddilutions at 37° C. for 1 h and 4° C. overnight. After washing,¹²⁵I-labeled mouse Guy's 13 was added and left to incubate for 2 h at37° C. The plates were washed again and the bound radioactivity wascounted in a gamma counter (Hydragamma 16, Innotec, GB). The results areexpressed as % inhibition of labeled mouse Guy's 13 binding, in which100% is the radioactive count from wells to which no blocking solutionhad been added.

d) Western Blot Analysis

Aliquots of 10 μl of leaf homogenates were boiled with 75 mM Tris-HCl(pH 6.8), 2% SDS, under reducing and nonreducing conditions. SDS-PAGE in10% acrylamide was performed, and the gels were blotted ontonitrocellulose. The blots were incubated for 16 h in TBS with 0.05%Tween 20 and 1% non-fat dry milk, followed by goat anti-mouse IgG1,kappa (Nordic Pharmaceuticals) or alpha chain-specific antisera (Sigma),and incubated for 2 h at 37° C. After washing, the second-layerantibody, an alkaline phosphatase-conjugated rabbit anti-goat IgG(Sigma) was applied for 2 hours at 37° C. Antibody binding was detectedby incubation with 300 μg/ml nitroblue tetrazolium and 15 p μg/ml5-bromo-4-chloro-3-idolyl phosphate (Promega).

e) DNA Sequencing

The DNA sequence of each cloned immunoglobulin gene insert confirmedthat no mutations had occurred during PCR amplification or the cloningprocedures. The introduction of the HindIII site in the λ/γ hybrid heavychains resulted in the predicted addition of the leucine residue betweenthe Cγ2 and Cα2 domains in Plant G2/A and leucine-lysine between the Cγ1and Cα2 domains in Plant G1/A. The additional Cγ2 domain in the PlantG2/A construct is predicted to increase the length of the heavy chain by141 amino acid residues (approximately 12000 Da). The plant G1/A heavychain in predicted to be slightly larger than the native Guy's 13 heavychain, by 33 amino acids, approximately 3000 Da.

Plasmid DNA that was purified from positive transformants in E. coli wassequenced. The immunoglobulin gene inserts were excised and sub-clonedinto Bluescript (Stratagene, USA). The DNA sequence was determined by adi-deoxy termination procedure (Sequenase, USB, USA).

f) Expression of Assembled Antibody

Western blot analysis on extracts from three representative F1 progenyplants was performed and reported in FIG. 2 of Ma et al., Eur. J.Immunol., 24:131-138 (1994). Samples run under reducing conditionsdemonstrate the presence of light (kappa) chain at approximately 25 Kd,in the mouse Guy's 13, as well as in the three transgenic plants, butnot in the control plant. Guy's 13 heavy (gamma) chain was also detectedin plant G13 at approximately 57 Kd, but not in the control plantextract. A single protein species was detected, unlike the hybridomaproducing the Guy's 13 antibody cell culture supernatant, in which a twoprotein species was a consistent finding. The difference in themolecular size of the mouse heavy chains is probably due toglycosylation differences, and the result suggests that in plants thetwo heavy chains may be glycosylated in the same way.

The heavy chains of plant G1/A and G2/A were detected with an anti-alphachain antiserum. Compared with the mouse Guy's 13 heavy chain,(approximately 57 Kd), the heavy chain of plant G1/A has a slightlyhigher relative molecular mass (approximately 60 Kd) and the plant G2/Aheavy chain is much larger (approximately 70 Kd). This is consistentwith the molecular weights predicted by sequence analysis. Several otherprotein species were detected in the transgenic plant extracts. Theseare likely to be proteolytic fragments of either light/heavy chaincomplexes, or of the heavy chain, as no bands were detected in theextract from the control transgenic plant. The anti-alpha chainantiserum did not cross-react with the mouse Guy's 13, which onlycontains gamma chain domains.

Samples were also run under nonreducing conditions to confirm theassembly of heavy and light chains into an immunoglobulin molecule andreported in FIG. 3 of Ma et al., Eur. J. Immunol., 24:131-138 (1994).Detection was with a labeled anti-kappa antiserum, and all threetransgenic plants had assembled immunoglobulin at the correct M_(r) ofabove 150 Kd for full-length antibody. The plant G13 antibody has thesame M_(r) as the mouse G13, but the plant G2/A and plant G1/Aantibodies have higher M_(r) as predicted. A number of smallerproteolytic fragments were also detected, which is consistent withprevious findings and the fact that a number of proteases are releasedby plants during the antibody extraction procedure. That these areantibody fragments, is confirmed by the absence of any detectable bandsin the control plant extract.

g) Antigen Binding

Ten plants which were producing immunoglobulin were made in total, andthe concentration of immunoglobulin in plant extracts varied between 1and 10 μg/ml (mean 4.5 μg/ml). For the murine antibody and therepresentative plants used in this study, the concentrations estimatedby ELISA were: mouse IgG-15.4 μg/ml, plant IgG-7.7 μg/ml, plant G1/A-1.5μg/ml and plant G2/A-2.1 μg/ml. The concentrations determined for plantantibodies containing hybrid heavy chains are possibly underestimated,as they do not carry all of the constant region determinants, ascompared with the standard mAb IgA used.

Titration curves for extracts from the three representative transgenicplants binding to SA I/II were generated and reported in FIG. 4 of Ma etal., Eur. J. Immunol., 24:131-138 (1994). Specific antibody wasdetectable in all three transgenic plant extracts, and the titrationcurves were similar to that of the murine hybridoma cell culturesupernatant, used at the same concentration. The binding of the plantG1/A antibody appeared to be slightly lower than the other antibodies,although the titration curve followed a similar pattern. No SA I/IIbinding activity was detected in the negative control plant nor didextracts from plants individually expressing light or heavy chains havebinding activity towards purified SA I/II. These findings demonstratethat the transgenic plants expressing both light and heavy chains haveassembled the antibody molecule correctly to form a functional antigenbinding site and that single light or heavy chains are not capable ofbinding the antigen.

The plant antibodies also recognized native antigen on the surface ofstreptococcal cells as shown in FIG. 5 of Ma et al., Eur. J. Immunol.,24:131-138 (1994) (S. mutans serotype c), which further confirms theintegrity of the antigen-binding site in the plant antibodies. Therewere no significant differences between the binding of the differentantibodies. Neither extracts from control plants, nor plants expressingonly heavy or light chains showed any binding to S. mutans cells. Therewas no binding to E coli cells by any of the plant extracts, atconcentrations of 1.0 and 0.5 μg/ml.

The plant antibodies competed with the original mouse Guy's 13 mbAb forbinding to SA I/II. Up to 85% inhibition of ¹²⁵I-labeled mouse Guy's 13mAb binding to SA I/II was demonstrated using the plant antibodies asshown in FIG. 6 of Ma et al., Eur. J. Immunol., 24:131-138 (1994). Asbefore, the inhibition titration curves of the plant antibodies weresimilar to each other, and comparable to that of the mouse Guy's 13,whereas the control plant extract gave no inhibition.

h) Aggregation of S. mutans

The action of the immunoglobulin produced in plants having the Guy's 13antigen binding region on bacteria was determined and reported in FIG. 7of Ma et al., Eur. J. Immunol., 24:131-138 (1994). Plant extracts weresterilized by filtration through a 0.22 μm pore size filter and dilutedtenfold with Todd Hewitt broth. The samples were inoculated with 0.05vol of an overnight S. mutans culture and incubated at 37° C. overnight.The samples were Gram stained and examined under oil immersionmicroscopy. S. mutans grown in the presence of mouse Guy's 13, plantGuy's 13, plant G1/A or plant G2/A became aggregated and cell clumpingwas evident. However, the control plant extract had no effect on S.mutans growth. None of the plant mAb appeared to affect S. mutans rateof growth, as determined by culture of viable organisms at 8, 12 and 16h. This result demonstrates not only that the plant antibodies havecorrectly assembled antigen-binding regions, but also that the antibodymolecules bind antigen bivalently.

Example 7 Production of Immunoglobulins Containing Protection Proteins

Four transgenic Nicotiana tabacum plants were generated to express (1) amurine monoclonal immunoglobulin kappa chain having the antigen bindingsite of the Guy's 13 light chain, (2) a hybrid IgA/G murineimmunoglobulin heavy chain containing Cγ and Cα chain domains and theantigen binding site of the Guy's 13 heavy chain, (3) a murine J chainand (4) protection protein comprised of amino acids 1-606 of rabbitpolyimmunoglobulin receptor and did not contain amino acids 627-675 ofthe rabbit polyimmunoglobulin receptor. See, Example 1. Successivesexual crosses between these plants resulted in simultaneous expressionof all four protein chains in the progeny plants. In some cases, backcrossing was used to produce homozygous plants. The four recombinantpolypeptides were assembled into a functional, high molecular weightimmunoglobulin containing a protection protein of approximately 470,000Kd. The assembly of the protection protein with the immunoglobulin wasdependent on the presence of a J chain, as no association of theprotection protein was detected when plants expressing antibody alonewere crossed with those expressing the protection protein. Microscopicevaluation of plants expressing the immunoglobulins containing theprotection protein demonstrated co-incident expression of protectionprotein and immunoglobulin heavy chains in single cells. Single cellsare able to produce immunoglobulin having a protection protein intransgenic plants, whereas two cells are required for natural productionof secretory immunoglobulin in mammals. The results demonstrate thatsexual crossing of transgenic plants expressing recombinant sub-units issuitable for large scale production of immunoglobulin containing aprotection protein for passive immunotherapy, as well as for expressingother complex protein molecules.

The immunoglobulin which contains the protection protein has the heavyand light chain antigen binding domains from the Guy's 13 monoclonalantibody that specifically recognize the cell surface adhesion moleculeSA 1/11 of an oral streptococcus as shown by Smith, R. & Lehner, T. OralMicrobiol. Immunol. 4, 153-158 (1989). Transgenic immunoglobulin of thistype containing only heavy and light chains has been generated inNicotiana tabacum plants as described in Example 6. A mouse J chainconstruct containing the coding length cDNA was amplified usingsynthetic oligonucleotide primers corresponding to the N terminus MKTHLLand the C terminus SCYPD of mouse J chain as described by Matsuuchi, L.,Cann, G. M. & Koshland, M. E. PNAS 83, 456-460 (1986). This amplifiednucleotide sequence was ligated into a constitutive plant expressionvector, pMON 530, that includes the 35S promoter from Cauliflower MosaicVirus and has been described by Rogers, S. G., Klee, H. J., Horsch, R.B. & Fraley, R. T. Meth. Enzymol. 153, 253-276 (1987). Tobacco leaftissue was transformed using agrobacterium containing the recombinantplasmid as described in the previous Examples. Regenerated plants werescreened for the production of messenger RNA encoding J chain andpositive transformants were self fertilized in order to generatehomozygous progeny. The J chain expressing plants were crossed initiallywith those expressing the chimeric immunoglobulin heavy chain and kappachain. Western blot analysis of the plant extract from plants expressingthe chimeric immunoglobulin heavy chain with anti-kappa antiserum undernon-reducing conditions, revealed a protein species of approximately 210Kd, which is consistent with the presence of the extra constant regiondomains present in the chimeric immunoglobulin heavy chain, as comparedwith the original IgG1 antibody. The progeny from the cross between theplant expressing the immunoglobulin and a J chain plant resulted in theappearance of a major immunoglobulin band at approximately twice therelative molecular mass of approximately 400 Kd, demonstrating thatassembly of the 3 polypeptides had occurred to form dimericimmunoglobulin (dlgA/G).

The protection protein construct consisted of a coding length cDNAamplified using synthetic oligonucleotide primers corresponding to the Nterminus MALFLL and AVQSAE at amino acids 601-606 of the C terminus ofrabbit polyimmunoglobulin receptor. The nucleotide sequence of therabbit polyimmunoglobulin receptor was reported by Mostov, K. E.,Friedlander, M. & Blobel, G. Nature 308, 37-43 (1984). The protectionprotein was generated in transgenic plants as described above andpositive transformants expressing the protection protein were identifiedby Western blot analysis.

Plants expressing J chain assembled with the immunoglobulin having theIgA/G heavy chains to form dimers were then crossed with a homozygousplant expressing the protection protein. The progeny plants expressingthe immunoglobulin having the protection protein contained a highermolecular weight protein species at approximately 470 Kd as determinedby Western blot analysis under non-reducing conditions. This molecularsize was consistent with that expected for an immunoglobulin containinga protection protein. This high molecular weight protein contained theprotection protein as confirmed by Western blotting, using antiserumthat specifically recognized the protection protein. The plant extractsalso contained a protein species of approximately 400 Kd correspondingto the dimers of IgA/G and a protein species of approximately 210 Kdcorresponding to the immunoglobulin with the chimeric heavy chain, butthese were only detected by anti-kappa antiserum and not theanti-protection protein antiserum. In the transgenic plant producing theprotection protein alone, there was no evidence that the protectionprotein assembled with endogenous plant proteins or formed multimers, asno high molecular weight proteins were detected in Western blottingunder non-reducing conditions. Western blot analysis demonstrated thatextracts from the plants expressing immunoglobulin heavy chain (IgA/G,dimeric IgA/G and the immunoglobulin containing a protection protein),but not the plants containing only the protection protein or J chain orwild-type plants, contained identical immunoglobulin derived heavy andlight chains. Furthermore, only the plants containing protectionproteins and the plants containing the IgG/A immunoglobulin having theprotection protein expressed proteins that were recognized by theantiserum that specifically recognized the protection protein. No crossreacting proteins were detected in extracts from the wildtype controlplant.

In mammals, the assembly of secretory component with the immunoglobulinrequires the presence of J chain as described by Brandtzaeg, P. & Prydz,H. Nature 311, 71-73 (1984). Plants expressing immunoglobulinscontaining a chimeric heavy chain (IgA/G) were crossed with plantsexpressing protection protein. None of the 10 resulting progeny thatexpressed immunoglobulin and the protection protein without J chainproduced assembled complexes as compared with the 10/10 plants thatco-expressed J chain dimerized immunoglobulin and the protection proteinwithout J chain, which assembled the M_(r) 470 Kd immunoglobulincontaining the protection protein. This confirms that J chain isrequired for the protection protein association with immunoglobulin asfound in mammals. Only the approximately 210 Kd monomeric form of theimmunoglobulin was recognized by anti-kappa antiserum, and the antiserathat specifically bound the protection protein, recognized freeprotection protein, but no immunoglobulin heavy or light chainsproteins.

Functional studies were carried out using the immunoglobulin produced inthe 5 plant constructs using ELISA. All plants expressing immunoglobulinlight and heavy chains, assembled functional immunoglobulin thatspecifically recognized streptococcal antigen (SA I/II). The levels ofbinding and titration curves were similar to those of the native mousehybridoma cell supernatant. No SA I/II binding was detected in plantsexpressing only J chain or only protection protein or in wildtypeplants. Binding of the immunoglobulins to immobilized purifiedstreptococcal antigen or to native antigen on the bacterial cell surfacewas also detected using the antiserum which specifically binds theprotection protein. In these assays, the binding of the immunoglobulincontaining the protection protein to the streptococcal antigen wasspecifically detected. These results confirmed that the protectionprotein was assembled with the immunoglobulin to produce animmunoglobulin containing a protection protein in a manner which did notinterfere with antigen binding.

The assembly of heavy and light chains into functional immunoglobulinmolecules in plants is very efficient as shown by Hiatt, A. C.,Cafferkey, R. & Bowdish, K. Nature 342, 76-78 (1989). A signal peptidemust be present on both heavy and light chain constructs to direct therecombinant proteins to the endoplasmic reticulum antibody for assemblyto take place in plants as was previously shown by Hiatt, A. C.,Cafferkey, R. & Bowdish, K. Nature 342, 76-78 (1989). This study hasdemonstrated the fidelity of immunoglobulin assembly which includesdimerization of monomeric antibody by J chain in the transgenic plants.These results demonstrated that in plants the dimeric immunoglobulinpopulation represents a major proportion (approx. 57%) of the totalantibody. These results also demonstrate the production of an assembledimmunoglobulin containing a protection protein which binds thecorresponding antigen as well as the parent murine monoclonal antibody,which makes up a major proportion of the total antibody when theprotection protein is incorporated (approximately 45%).

Co-expression of dimeric immunoglobulin with the protection protein inplants has led to assembly of a functional immunoglobulin containing aprotection protein. All four transgenes for this complex protein wereintroduced into plants with the identical pMON530 expression cassetteand native leader sequences. This vector contains a promoter sequencederived from the 35S transcript of the cauliflower mosaic virus whichdirects expression of transgenes in a variety of cell types of mostplant organs as has been described by Benfey, P. N. & Chua, N-H. Science250, 959-966 (1990); and Barnes, W. M. PNVAS 87, 9183-9187 (1990).Directing expression of all four transgenes with the same promotermaximized the likelihood of coincidental expression in a common plantcell. Microscopic observation of plants expressing an immunoglobulincontaining a protection protein revealed that many cell types of theleaves contain the individual protein components that make up theimmunoglobulin. These proteins accumulated at highest concentration inbundle sheath cells and were confined by the cell walls of these andother cells, but were not found in intercellular spaces. Restriction ofthe largest immunoglobulin components, the protection protein and thechimeric immunoglobulin heavy chain, within the confines of aprotoplastic or apoplastic compartment of individual cells wouldconstrain the assembly of the secretory immunoglobulin to those cells inwhich all the component molecules are synthesized. The subcellularsite(s) and mechanism of assembly remain to be determined, assembly ofIgG heterotetramers in plants requires targeting of both proteins to theendomembrane system as has been previously shown by Hiatt, A. C.,Cafferkey, R. & Bowdish, K. Nature 342, 76-78 (1989); and Hein, M. B.,Tang, Y., McLeod, D. A., Janda, K. D. & Matt, A. C. Biotechnol Prog. 7,455-461 (1991).

In addition, we have demonstrated that a protection protein derived frommature secretory component devoid of signals for membrane integration,transcytosis or subsequent proteolysis can be assembled with chimericimmunoglobulin heavy chain containing immunoglobulin gamma and alphaprotein domains. These results demonstrate that the inherent functionsof IgG constant regions (protein A binding, complement fixation, Fcreceptor activity) may be maintained in a dimeric immunoglobulin,capable of binding to a protective protein. These additionalcapabilities may be employed to enhance the function of animmunoglobulin used for passive immunotherapy and the development ofplants capable of generating a functional immunoglobulin containing aprotection protein will have significant implications in passiveimmunotherapy. The level of expression of the immunoglobulin containinga protection protein is high and the production can be scaled up toagricultural proportions, to allow economical production of monoclonalantibodies.

Methods

The following methods were used to prepare and analyze theImmunoglobulin of this Example.

i) Antibody Assembly in Transgenic Nicotiana tabacum.

Leaf segments were homogenized in 150 mM NaCl 20 mM Tris-HCl (pH8)(TBS), with leupeptin (10 μg/ml). The extracts were boiled for 3minutes, in 75 mM Tris-HCl (pH6.8), 2% SDS, under non-reducingconditions and SDS-PAGE in 4% acrylamide was performed. The gels wereblotted onto nitrocellulose. The blots were incubated for 2 hrs in TBSwith 0.05% Tween 20 and 1% non-fat dry milk, followed by the appropriateantiserum and incubated for 2 hrs at 37° C. After washing, the secondlayer alkaline phosphatase conjugated antibody was applied for 2 hrs at37° C. Antibody binding was detected by incubation with 300 mg/mlnitroblue tetrazolium and 150 mg/ml 5-bromo-4-chloro 3-indolylphosphate.

These extracts were analyzed using western analysis to determine whetherthe immunoglobulins were assembled into immunoglobulin molecules byanalyzing Western blots of plant extracts prepared under non-reducingconditions, were with anti-kappa antiserum (Bradsure, UK) and anantiserum which specifically recognizes protection protein. Theimmunoglobulins produced in the plants were compared to the monoclonalIgG1 Guys 13 immunoglobulin described by Smith, R. & Lehner, T. OralMicrobiol. Immunol. 4, 153-158 (1989).

ii) Western Analysis.

Western analysis was performed on each of the plant extracts preparedunder reducing conditions to identify individual protein components ofthe immunoglobulin. Samples of the various plant extracts were preparedas described previously, but with the addition of 5% β-mercaptoethanol.SDS-PAGE in 10% acrylamide was performed and the protein in the gelstransferred to nitrocellulose. Individual proteins were detected usinganti-mouse γ1 heavy chain (Sigma, UK); anti-mouse kappa chain (Bradsure,UK); or an antiserum that specifically recognized the protectionprotein, followed by the appropriate alkaline phosphatase conjugatedantibody.

iii) Western Analysis to Show Production of Immunoglobulin Having aProtection Protein

Western analysis of transgenic plant extract was performed as describedin ii) above. The plant extracts from plants expressing theimmunoglobulin containing the protection protein were subjected toSDS-PAGE under both non-reducing and reducing conditions and theproteins transferred to nitrocellulose. The immunoglobulin componentswere detected with an anti-kappa antiserum or with a sheep antiserumwhich specifically recognized the protection protein followed by anappropriate alkaline phosphatase labeled 2° antibody.

iv) Expression of Antigen-Specific Immunoglobulin Containing aProtection Protein in Transgenic Nicotiana tabacum.

To demonstrate that the plants were producing antigen-specificimmunoglobulin, plant extract binding to purified streptococcal antigen(SA) I/II, detected with horseradish peroxidase labeled anti-kappa chainantiserum was determined. The presence of a protection protein in theantigen-specific immunoglobulin was demonstrated by plant extractbinding to purified streptococcal antigen I/II and streptococcal cellsdetected with a sheep antiserum immunospecific for a protection protein,followed by alkaline phosphatase labeled donkey anti-sheep antiserum.These tests for antigen-specific immunoglobulin were carried out inmicrotitre plates that were coated with purified SA I/II (2 μg/ml) inTBS, or log phase growth Strep, mutans (NCTC 10449), in bicarbonatebuffer (pH 9.8). Blocking was with 5% non-fat dry milk in TBS at roomtemperature for 2 hours. Plant leaves were homogenized in TBS with 10μg/ml leupeptin (Calbiochem, USA). Mouse Guy's 13 hybridoma cell culturesupernatant (IgG) was used as a positive control. The supernatants wereadded in serial two-fold dilutions to the microtitre plate andincubation was at room temperature for 2 hours. After washing with TBSwith 0.05% Tween 20, bound immunoglobulin chains were detected witheither a goat anti-mouse light chain specific antibody, conjugated withhorseradish peroxidase (Nordic Pharmaceuticals, UK), or a sheep anti-SCantiserum, followed by an alkaline phosphatase labeled donkey anti-sheepantibody for 2 hours at room temperature. Detection was with2,2′-azino-di-[3-ethyl-benzthiazolin-sulphonate (Boehringer, W. Germany)for HRPO conjugated antibody or disodium p-nitrophenyl phosphate (Sigma,UK) for alkaline phosphatase conjugated antibody.

v) Localization of Immunoglobulin Components in Plants

Photomicrographs of transgenic plants expressing immunoglobulinscontaining protection proteins and control Nicotiana tabacum leaf wereprepared using immunogold detection of murine alpha chain. Briefly, leafblades were cut into 2 mm×10 mm segments and fixed in 3% (w/v)paraformaldehyde, 0.5% (w/v) glutaraldehyde, 5% (w/v) sucrose in 100 mMsodium phosphate (pH 7.4). After dehydration in anhydrous ethanol, leafsegments were infiltrated with xylene, embedded in paraffin and cut into3 mm sections and mounted on glass slides for immunochemical staining.The leaf sections were incubated with primary antibodies, affinitypurified rabbit anti-mouse alpha chain (which reacts with the A/G hybridheavy chain) or sheep anti-rabbit SC, and then with secondary antibody;goat anti-rabbit-10 mn gold or rabbit anti-sheep-10 mn gold. Theimmunogold signal was intensified by silver enhancement. The plants werevisualized using both Phase contrast and bright field microscopy on thesame leaf cross section. Immunolocalization of the protection protein onserial sections was used to show the same cellular localization forheavy chain as immunoglobulin. The analysis was carried out on thefollowing cells and cell compartments: spongy mesophyll cells, epidermalcells, intercellular spaces, palisade parenchyma cells, and vascularbundles.

Further analysis of the exact localization of immunoglobulin componentswas carried out by analyzing serial sections of Nicotiana tabacumvascular bundle and control Nicotiana tabacum vascular bundle withimmunogold detection for each of the components of the immunoglobulin.Serial sections of a transgenic plant leaves from plants expressingsecretory immunoglobulin were incubated with an antibody thatspecifically recognizes the protection protein or with anti-IgA antibodyfollowed by the appropriate gold-labeled secondary antibody. A controlleaf section from a transgenic plant that did not contain anyimmunoglobulin coding sequences was also incubated with anti-IgAantibody, followed by gold-labeled goat anti-rabbit antiserum, or withthe gold-labeled secondary antibodies alone and confirmed thespecificity of staining. Both Phase contrast illumination of a minorvascular bundle and Bright field illumination of the same field wereused to show immunogold localization of the protection protein. Brightfield illumination of a serial leaf cross section of the vascular bundledemonstrated the same immunogold localization of the immunoglobulinheavy chain as was shown for the protection protein.

Example 8 Production of a Useful Plant Extract ContainingImmunoglobulins Having a Protection Protein

Plant pieces (either leaf, stem, flower, root, or combinations) fromplants producing immunoglobulins containing a protection protein weremixed with homogenization buffer (2 milliliter buffer per gram of plantmaterial; homogenization buffer: 150 mM NaCl, 20 mM Tris-Cl, pH 7.5),homogenized into a pulp using a Waring blender and centrifuged at10,000×g to remove debris. The supernatant was then extracted with anequal volume of HPLC-grade ethyl acetate by shaking at room temperature,followed by centrifugation at 10,000×g. The aqueous phase wastransferred to another container, remaining ethyl acetate was removedfrom the aqueous phase by placing the solution under vacuum. Theresulting crude extract consistently contained 100 μg immunoglobulinhaving a protection protein per ml. This method is useful for any plantcontaining an immunoglobulin having a protection protein.

A number of methods for homogenization have been used including a mortarand pestle or a Polytron and can be performed either in the cold or atroom temperature.

The extract may be further purified by delipidation, by extraction withhexane or other organic solvents. Delipidation is not essential forderiving a useful product from the plant extract but is advantageous incases where the final product is a purified immunoglobulin having aprotection protein. In many instances the crude extract will contain asufficiently high quantity of immunoglobulin having a protection protein(i.e. 100 μg/mL) to be useful without any further purification orenrichment. For an oral application, the extract would be mixed withcommonly used flavorings and stabilizers. For a dental application, theextract would in addition be mixed with a gelling reagent to maintaincontact of the extract with teeth. For a gastric application, theflavored extract could be swallowed directly.

Example 9 Stability of an Immunoglobulin Containing a Protection Protein

Two sets of crude plant extracts were prepared as described above. Thefirst extract was derived from a plant expressing an IgG1 antibody andthe second extract was derived from a plant expressing an immunoglobulincontaining a protection protein. Crude plant extracts of this type fromplants are known to contain a variety of proteolytic enzymes. Prolongedincubation of extracts at room temperature or at 37° C. thereforeconstitutes a proteolytic digestion.

Using ELISA the quantity of gamma-kappa complexes in the two extractswas determined as a function of time at both room temperature and 37° C.In these assays, an anti-kappa chain antibody was used to coat the platefollowed by incubation with the plant extract at 37° C. for 1 hour. Ananti-gamma chain antibody conjugated to HRPO was used for detection ofimmunoglobulin derived from the plant. The quantity of immunoglobulinhaving a protection protein contained in the extract immediately afterthe extract was prepared was taken to be 100%. After 3 hours at roomtemperature, the IgG1 contained 40% and the immunoglobulin containingthe protection protein contained >95%. After 6 hours, the remaining IgG1antibody was 20% and the immunoglobulin containing the protectionprotein abundance was still >95%. After 12 hours, there was nodetectable IgG1 whereas ˜90% of the immunoglobulin containing theprotection protein remained. A significant decrease (to ˜70%) in theabundance of protected antibody was not observed until 48 hours afterthe extract was prepared.

Example 10 Eukaryotic Tetratransgenic Cells Expressing ImmunoglobulinsContaining Protection Protein

The four chains comprising the immunoglobulin containing a protectionprotein can also be expressed in other cell types either in in vitro(cell cultures) or in vivo (transgenic animals). See, Manipulating theMouse Embryo; A Laboratory Manual, B. Hogan et al., Cold Spring HarborLaboratory (1986). In the case of transgenic animals, purifiedpreparations of appropriate vector DNAs are adjusted to a finalconcentration of 2 ng/μl in 10 mM Tris, 0.2 mM EDTA, pH 7.4. Pronuclearinjections are performed using zygotes prepared from inbred animals.Injected eggs are then transferred to pseudopregnant females usingstandard techniques. Live born animals are then screened for thepresence of transgenes using any of a number of commonly used techniquessuch as PCR and ELISA. Members of the pedigree expressing differentcomponents of the immunoglobulin containing the protection protein arethen mated to produce multi-transgene animals. Progeny from thesecrosses are then screened to identify those that express all fourchains. Depending on the type of vector used for zygotic injectionsvarious cell types can be identified in the transgenic animals whichassemble the complete immunoglobulin containing a protection protein.These vector DNAs can consist of specific promoter elements which allowtranscription of the transgene in particular cell types or tissues. Eachvector could express a single component of the protected antibody(IgG/A, J chain, protection protein, or kappa chain) or couldpotentially express more than one component. In this instance, thevector would contain an appropriate number of promoter regions andrestriction sites to allow for transcription of each transgene.

Expression of all four chains in a cell culture system can be achievedusing a DNA vector from which each component can be individuallypromoted. This would require four expression cassettes (containingpromoter, multiple cloning site, and polyadenylation region) on the samevector DNA. Alternatively, individual cell lines can be sequentiallytransfected with individual vectors expressing single chains so long aseach vector confers a selective resistance onto the cell line.

Commonly available vectors, such as pMAMneo (Clontech) can be adaptedeither for multiple expression or as a series of vectors expressingdistinct selectable markers.

Transfection of any eukaryotic cells, such as fibroblasts, is done byconventional techniques. Briefly, cells are split 1:20 the day beforetransfection and are transfected at approximately 30% confluency using125 mM CaCl2, 140 mM NaCl, 25 mM Hepes, 0.75 mM NaHPO4, pH 7.05, and 5μg DNA/10 cm dish. After 16 hours of DNA incubation, cells are shockedby 10% dimethyl sulfoxide for 3 minutes. Forty eight hours aftertransfection, cells are subjected to selection by growth in theappropriate medium containing an antibiotic or other cytotoxic reagent.

The resulting cells produce all the components for the immunoglobulincontaining the protection protein. These components are properlyassembled to produce a functional immunoglobulin containing a protectionprotein.

Example 11 Engineering a Protection Protein Fused to a Portion of theCytoplasmic Domain of the Rabbit Polyimmunoglobulin Receptor

The construction of DNA segments encoding a protection protein fused toa segment encoding a segment of the cytoplasmic domain of the rabbitpolyimmunoglobulin receptor is produced as follows. Protection proteincDNA encoding from the first amino acid of the signal sequence(MET_(—18)) to GLU₆₀₆ is ligated into any plant expression vector, suchas the pMON530 vector (digested with Bgl II and Xho I) as a Bgl II-Xho Ifragment. This protection protein derivative is obtained by PCRamplification using the appropriate oligonucleotide primers containingeither a Bgl II or Xho I recognition sequence which are alsocomplementary to DNA encoding residues −18 to −13 and residues 601 to606 of the rabbit polyimmunoglobulin receptor respectively. The sameprocedure is performed in order to obtain a protection protein cDNAencoding from MET_(—18) to ALA₆₂₈ except that the oligonucleotidecontaining an Xho site is also complementary to the protection proteincDNA encoding residues 623 to 628.

The cDNA encoding the rabbit polyimmunoglobulin receptor cytoplasmicdomain fragment is obtained, also by PCR amplification, as a Xho Ifragment. The oligonucleotides employed are complementary to DNAencoding from ARG₆₅₃ to ALA₇₅₅ both containing Xho I recognitionsequences. This fragment is then ligated into the pMON530 vectors whichcontain the either of the protection protein cDNAs described above. Theappropriate orientation of the cytoplasmic domain cDNA is determined byrestriction digestions and by sequence analysis of plasmids obtainedfrom transformed bacterial colonies.

The oligonucleotides employed for PCR amplification contain theappropriate number of nucleotides to ensure that the resulting cDNAs arein frame and capable of being translated as a continuous fusion proteincontaining both protection protein and cytoplasmic domain.

The resulting constructs in the appropriate orientation encode aprotection protein fused directly to the polyimmunoglobulin receptorcytoplasmic domain with no functional transmembrane segment, operablylinked to a DNA segment (promoter) enabling expression in a plant cell.The constructs encode two additional amino acids (SER-TRP) which arederived from introduction of the Xho I restriction site and which serveas a linker between the protection protein and the cytoplasmic domain.

These vectors are then used to transform Agrobacterium as previouslydescribed which in turn is used to transform plant cells. The sametechniques described in the above Examples are used to produce a plantexpressing this protein as part of an immunoglobulin.

1-94. (canceled)
 95. An immunoglobulin comprising a protection proteinin association with an immunoglobulin derived heavy chain having atleast a portion of an antigen binding domain.
 96. The immunoglobulin ofclaim 95 further comprising an immunoglobulin derived light chain havingat least a portion of an antigen binding domain associated with saidimmunoglobulin derived heavy chain.
 97. The immunoglobulin of claim 95further comprising a second immunoglobulin derived heavy chain having atleast a portion of an antigen binding domain associated with saidprotection protein.
 98. The immunoglobulin of claim 97 furthercomprising at least one immunoglobulin derived light chain having atleast a portion of an antigen binding domain bound to said secondimmunoglobulin derived heavy chain.
 99. The immunoglobulin of claim 95further comprising an immunoglobulin J chain bound to saidimmunoglobulin derived heavy chain and optionally to a secondimmunoglobulin derived heavy chain.
 100. The immunoglobulin of claim 95that is a therapeutic immunoglobulin.
 101. The immunoglobulin of claim100 wherein said therapeutic immunoglobulin binds to mucosal pathogenantigens.
 102. The immunoglobulin of claim 101 that is capable ofpreventing dental caries.
 103. The immunoglobulin of claim 95 whereinsaid antigen binding domain is capable of binding an antigen from S.mutans serotypes c, e and f or S. sobrinus serotypes d and g.
 104. Theimmunoglobulin of claim 95 wherein said protection protein has an aminoacid sequence which substantially corresponds to at least a portion ofthe amino acid residues selected from the group consisting of 1 to 627and 1 to 606 of the rabbit polyimmunoglobulin receptor and wherein saidprotection protein does not have an amino acid residue sequencecorresponding to amino acid residues 628-755 of the rabbitpolyimmunoglobulin receptor.
 105. The immunoglobulin of claim 104wherein said protection protein has an amino acid sequence which doesnot contain amino acid residues corresponding to amino acid residues 628to 775 of the rabbit polyimmunoglobulin receptor and which does containamino acid residues which correspond to one or more of the followingamino acid segments: a) amino acids corresponding to amino acid residues21-43 of the rabbit polyimmunoglobulin receptor; b) amino acidscorresponding to amino acid residues 1-118 of the rabbitpolyimmunoglobulin receptor; c) amino acids corresponding to amino acidresidues 119-223 of the rabbit polyimmunoglobulin receptor; d) aminoacids corresponding to amino acid residues 224-332 of the rabbitpolyimmunoglobulin receptor; e) amino acids corresponding to amino acidresidues 333-441 of the rabbit polyimmunoglobulin receptor; f) aminoacids corresponding to amino acid residues 442-552 of the rabbitpolyimmunoglobulin receptor; g) amino acids corresponding to amino acidresidues 553-606 or 553-627 of the rabbit polyimmunoglobulin receptor.106. The immunoglobulin of claim 95 wherein said protection protein hasan amino acid sequence which does not contain amino acid residues of apolyimmunoglobulin receptor of a species which are analogous to aminoacid residues 628 to 755 of the rabbit polyimmunoglobulin receptor andwhich does contain amino acid residues from a polyimmunoglobulinreceptor of a species which are analogous to one or more of thefollowing amino acid segments: a) amino acids corresponding to aminoacid residues 21-43 of the rabbit polyimmunoglobulin receptor; b) aminoacids corresponding to amino acid residues 1-118 of the rabbitpolyimmunoglobulin receptor; c) amino acids corresponding to amino acidresidues 119-223 of the rabbit polyimmunoglobulin receptor; d) aminoacids corresponding to amino acid residues 224-332 of the rabbitpolyimmunoglobulin receptor; e) amino acids corresponding to amino acidresidues 333-441 of the rabbit polyimmunoglobulin receptor; f) aminoacids corresponding to amino acid residues 442-552 of the rabbitpolyimmunoglobulin receptor; g) amino acids corresponding to amino acidresidues 553-606 or 553-627 of the rabbit polyimmunoglobulin receptor.107. The immunoglobulin of claim 106 wherein said species is human. 108.The immunoglobulin of claim 95 wherein said protection protein includesthe amino acid sequence of at least one of the domains selected from thegroup consisting of the following portions of the rabbitpolyimmunoglobulin receptor: domain I, domain II, domain III, domain IV,domain V, and amino acid residues 553 to 627 of domain VI; and does nothave an amino acid sequence corresponding to amino acid residues 628-755of the rabbit polyimmunoglobulin receptor.
 109. The immunoglobulin ofclaim 95 wherein said protection protein does not have any amino acidsequence which corresponds to or is analogous to amino acid residues628-755 of the rabbit polyimmunoglobulin receptor and which doesinclude: a) at least one domain which is from the polyimmunoglobulinreceptor of a first animal and which is analogous to at least a portionof the following amino acid segments of the rabbit polyimmunoglobulinreceptor: domain I, domain II, domain III, domain IV, domain V, andamino acid residues 553 to 627 of domain VI; b) at least one domainwhich is from the polyimmunoglobulin receptor of a second animal andwhich corresponds to or is analogous to the following amino acid residuesegments of the rabbit polyimmunoglobulin receptor: domain I, domain II,domain III, domain IV, domain V, and amino acid residues 553 to 627 ofdomain VI.
 110. The immunoglobulin of claim 95 wherein said protectionprotein does not have any amino acid sequence which corresponds to or isanalogous to amino acid residues 628-755 of the rabbitpolyimmunoglobulin receptor and which does include: a) at least oneamino acid segment which is from the polyimmunoglobulin receptor of afirst animal and which is analogous to at least a portion of thefollowing amino acid residue segments of the rabbit polyimmunoglobulinreceptor: domain I, domain II, domain III, domain IV, domain V, andamino acid residues 553 to 627 of domain VI; b) at least one amino acidsegment which is from the polyimmunoglobulin receptor of a second animaland which corresponds to or is analogous to the following amino acidresidue segments of the rabbit polyimmunoglobulin receptor: domain I,domain II, domain III, domain IV, domain V, and amino acid residues 553to 627 of domain VI.
 111. The immunoglobulin of claim 110 wherein saidfirst animal is a mammal and said second animal is a rabbit.
 112. Theimmunoglobulin of claim 110 wherein said first animal is a human andsaid second animal is a rabbit.
 113. The immunoglobulin of claim 95wherein said immunoglobulin derived heavy chain contains at least aportion of an IgM or IgA heavy chain of any subtype.
 114. Theimmunoglobulin of claim 95 wherein said immunoglobulin derived heavychain is comprised of immunoglobulin domains from two different isotopesof immunoglobulin.
 115. The immunoglobulin of claim 114 wherein saidimmunoglobulin domains are selected from the group consisting of: a) theC_(H)1 of a mouse IgG1 and the C_(H)2 and C_(H)3 of mouse IgA; and b)the C_(H)1 and C_(H)2 of a mouse IgG1 and the C_(H)2 and C_(H)3 of mouseIgA;
 116. The immunoglobulin of claim 95 wherein said antigen bindingdomain substantially corresponds to the Guy's 13 heavy chain variableregion.
 117. The immunoglobulin of claim 96 wherein said antigen bindingdomain substantially corresponds to the Guy's 13 light chain variableregion.
 118. A composition comprising the immunoglobulin of claim 95 andat least one plant macromolecule.
 119. The composition of claim 118wherein said plant macromolecule is derived from a dicotyledonous,monocotyledonous, solanaceous, alfalfa or tobacco plant.
 120. Thecomposition of claim 118 wherein said plant macromolecule is selectedfrom the group consisting of ribulose bisphosphate carboxylase, lightharvesting complex, pigments, secondary metabolites and chlorophyll.121. The composition of claim 118 wherein said immunoglobulin is presentin a concentration of between 0.001% and 99% mass excluding water. 122.The composition of claim 119 wherein said plant macromolecules arepresent in a concentration of between 1% and 99% mass excluding water.123. A method of producing the immunoglobulin of claim 95 comprising thesteps of: (a) introducing into a plant cell an expression vectorcontaining a nucleotide sequence encoding a protection protein operablylinked to a transcriptional promoter; and (b) introducing into saidplant cell an expression vector containing a nucleotide sequenceencoding an immunoglobulin derived heavy chain having at least a portionof an antigen binding domain operably linked to a transcriptionalpromoter.
 124. The method of claim 123 further comprising the step of:(c) introducing into said plant cell an expression vector containing anucleotide sequence encoding an immunoglobulin derived light chainhaving at least a portion of an antigen binding domain operably linkedto a transcriptional promoter.
 125. The method of claim 123 furthercomprising the step of introducing into said plant cell an expressionvector containing a nucleotide sequence encoding an immunoglobulin Jchain operably linked to a transcriptional promoter.
 126. The method ofclaim 123 wherein said immunoglobulin derived heavy chain isimmunoglobulin alpha chain and said immunoglobulin derived light chainis an immunoglobulin kappa or lambda chain.
 127. The method of claim 123wherein said immunoglobulin derived heavy chain is comprised of portionsof immunoglobulin alpha chain and immunoglobulin gamma chain.
 128. Themethod of claim 123 wherein said plant cells are part of a plant. 129.The method of claim 123 further comprising growing said plant cells intoa regenerated plant.
 130. The method of claim 128 wherein said plant isa dicotyledonous, monocotyledonous, solanaceous, leguminous, alfalfa ortobacco plant.
 131. The method of claim 123 wherein said immunoglobulinderived heavy chain is a chimeric immunoglobulin heavy chain.
 132. Amethod of producing a therapeutic immunoglobulin composition containingplant macromolecules, said method comprising the step of shearing underpressure plants or parts thereof to produce a pulp containing atherapeutic immunoglobulin and plant macromolecule mixture, saidimmunoglobulin comprising a protection protein, and wherein saidimmunoglobulin is encoded by at least one nucleic acid sequence that hasbeen introduced into the cells of said plants.
 133. The method of claim132 further comprising the step of separating said solid plant derivedmaterial from said liquid.
 134. The method of claim 132 wherein saidportion of said plant is a leaf, stem, root, tuber, fruit or entireplant.
 135. The method of claim 132 wherein said shearing isaccomplished by a mechanical device which releases liquid from theapoplast or symplast of said plant.
 136. The method of claim 133 whereinsaid separation is by centrifugation, settling, flocculation orfiltration.
 137. A method for producing an assembled immunoglobulinmolecule having heavy, light and J chains and a protection proteincomprising the steps of: a) introducing into a eukaryotic cellnucleotide sequences operably linked for expression encoding: i) animmunoglobulin derived heavy chain having at least a portion of anantigen binding domain, ii) an immunoglobulin derived light chain havingat least a portion of an antigen binding domain, iii) an immunoglobulinJ chain, and iv) a protection protein; and b) maintaining said cellunder conditions allowing production and assembly of said immunoglobulinderived heavy and light chains, said immunoglobulin J chain and saidprotection protein into an immunoglobulin molecule.
 138. A method forproducing an assembled immunoglobulin molecule having heavy, light and Jchains and a protection protein by maintaining under conditions allowingprotein production and immunoglobulin assembly, a eukaryotic cellcontaining nucleotide sequences operably linked for expression encoding:i) an immunoglobulin derived heavy chain having at least a portion of anantigen binding domain, ii) an immunoglobulin derived light chain havingat least a portion of an antigen binding domain, iii) an immunoglobulinJ chain, and iv) a protection protein.
 139. The method of claim 137wherein said eukaryotic cell is a plant cell.
 140. A method of making animmunoglobulin resistant to environmental conditions comprising thesteps of: a) operably linking a nucleotide sequence encoding at least aportion of the antigen binding domain derived from an immunoglobulinheavy chain to a nucleotide sequence encoding at least one domainderived from an immunoglobulin alpha heavy chain to form a nucleotidesequence encoding a chimeric immunoglobulin heavy chain; b) expressingsaid nucleotide sequence encoding said chimeric immunoglobulin heavychain to produce said chimeric immunoglobulin heavy chain in a plantcell which also contains at least one other molecule selected from thegroup consisting of: a protection protein, an immunoglobulin derivedlight chain having at least a portion of an antigen binding domain andan immunoglobulin J chain; and thereby allowing the chimericimmunoglobulin heavy chain to assemble with said at least one othermolecule to form said immunoglobulin resistant to said environmentalconditions.
 141. The method of claim 140 wherein said other molecule isa protection protein and said plant cell also contains an immunoglobulinderived light chain having at least a portion of an antigen bindingdomain and an immunoglobulin J chain.
 142. A process for producing animmunoglobulin resistant to environmental conditions by maintainingunder conditions allowing protein production and immunoglobulin assemblya plant cell containing: a) a nucleotide sequence encoding a chimericimmunoglobulin heavy chain in which a nucleotide sequence encoding atleast a portion of an antigen binding domain derived from heavy chain isoperably linked to a nucleotide sequence encoding at least one domainderived from an immunoglobulin alpha heavy chain; and b) at least oneother molecule selected from the group consisting of: a protectionprotein, an immunoglobulin derived light chain having at least a portionof an antigen binding domain and an immunoglobulin J chain; therebyallowing the chimeric immunoglobulin heavy chain to assemble with saidat least one other molecule to form said immunoglobulin resistant tosaid environmental conditions.
 143. The immunoglobulin of claim 95wherein said chimeric immunoglobulin heavy chain contains animmunoglobulin domain from one of the following immunoglobulin heavychains: IgG, IgA, IgM, IgE, IgD; and also contains a protectionprotein-binding domain from IgA or IgM.
 144. The immunoglobulin of claim143 wherein said immunoglobulin heavy chains are human, rodent, rabbit,bovine, ovine, caprine, fowl, canine, feline or primate immunoglobulinheavy chains.
 145. The immunoglobulin of claim 143 wherein saidprotection protein-binding domain is from the IgA of a human, rodent,rabbit, bovine, ovine, canine, feline or primate.
 146. Theimmunoglobulin of claim 143 wherein said chimeric immunoglobulin heavychain is comprised of immunoglobulin chains of mouse IgG1 and saidprotection protein-binding domain is from mouse IgA or IgM.
 147. Theimmunoglobulin of claim 143 wherein said chimeric immunoglobulin heavychain is comprised of immunoglobulin domains of a human IgG, IgM, IgD orIgE and said protection protein-binding domain is from a human IgA orIgM.